Protein activity modification

ABSTRACT

A method of modifying tissue behavior, comprising:
         determining a desired modification of tissue behavior for at least one of treatment of a disease, short or long term modification of tissue behavior, assessing tissue state and assessing tissue response to stimulation;   selecting an electric field having an expected effect of modifying protein activity of at least one protein as an immediate response of a tissue to the field, said expected effect correlated with said desired modification; and   applying said field to said tissue.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/919,491 filed Mar. 8, 2009, now U.S. Pat. No. 8,548,583, which is aNational Phase of PCT Patent Application No. PCT/US2006/017281, filedMay 4, 2006, published as WO/2006/119467 which is a Continuation-in-Part(CIP) of PCT Patent Application No. PCT/US2005/044557 filed Dec. 9,2005, published as WO/2006/073671, which is a Continuation-in-Part (CIP)of PCT Patent Application No. PCT/US2004/007589 filed Mar. 10, 2004,published as WO/2004/08053 and claims the benefit of priority under 35USC §119(e) of U.S. Provisional Patent Application Nos. 60/634,625 filedDec. 9, 2004, 60/677,761 filed May 4, 2005 and 60/719,517 filed Sep. 22,2005.

PCT Patent Application No. PCT/US2006/017281 is also a continuation ofPCT Patent Application No. PCT/IL2006/000204 filed Feb. 16, 2006,published as WO/2006/087717A2.

This application is also related to PCT Patent Application Nos.PCT/IL97/00012 filed Jan. 8, 1997, published as WO/1997/025098A1 andPCT/IB00/01523 filed Oct. 4, 2000, published as WO/2001/024871A2.

The disclosures of all these above applications are incorporated hereinby reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to modification of tissue behavior, forexample using electric fields and for example using biochemical markersas feedback.

While some proteins have a mainly structural role in cellular life, manyproteins are biologically active. Living cells include many mechanismsby which the biological activity of a protein is modulated, including:modification of concentration of the protein or its substrates,modification of the concentration of materials that catalyzes proteinactivity, indirect modification of protein structure, such as bychanging of pH or concentrations of materials that modify proteinstructure, and direct modification of protein spatial structure and/orcharge distribution by attachment of cofactors such as a phosphatemoiety (phosphorylation), glucose, ions, metal ions, heme groups oriron-sulfur complexes and coenzymes for example.

The symptoms of many diseases include changes in protein activity, asindicated, for example, by phosphorylation (hyper- or hypo-). Oneexample is cardiac heart failure, where, as the disease progresses thephosphorylation of some proteins goes down and others go up. Levels ofvarious proteins also change.

As described, for example in N Engl J Med 346:1357, 2002, the disclosureof which is incorporated herein by reference, patients with CHF whorespond to therapy with beta blockers manifest reversal that isnormalization of the maladaptive fetal gene program.

In a paper entitled “Voltage-dependent potentiation of the activity ofcardiac L-type calcium channel al subunits due to phosphorylation bycAMP-dependent protein kinese”, by Adrian SCULPTOREANU, Eric ROTMAN,Masami TAKAHASHI, Todd SCHEUER, AND William A. CATTERALL, in Proc. Natl.Acad. Sci. USA Vol. 90, pp. 10135-10139, November 1993 (Physiology), thedisclosure of which is incorporated herein by reference, fastphosphorylation of trans-membrane calcium channels and a possiblemechanism therefore, are described.

U.S. Pat. No. 6,919,205, the disclosure of which is incorporated hereinby reference, describes regulation of type II cartilage genes andproteins using electromagnetic and electric fields.

PCT publication WO 2005/056111, the disclosure of which is incorporatedherein by reference describes using a PMF signal on calcium dependentmyosin phosphorylation in a cell free reaction mixture.

PCT publication WO 2005/102188, the disclosure of which is incorporatedherein by reference, describes PMF stimulation applied to Jurkat cellsreduces DNA synthesis and makes them behave like normal T-lymphocytesstimulated by antigens at the T-cell receptor such as anti-CD3, possiblyby interacting with the T-cell receptor.

PCT publication WO 2005/105013, the disclosure of which is incorporatedherein by reference, describes applying a PMF to a heart in order toachieve angiogenesis and neovascularization.

SUMMARY OF THE INVENTION

A broad aspect of some embodiments of the invention relates to modifyingthe activity of proteins or other biochemicals optionally in situ and/orin vivo, for example, by modifying protein phosphorylation, usingelectro-magnetic or electrostatic fields. In an exemplary embodiment ofthe invention, the activity of the protein that is modified is one ormore of signaling, catalysis, material transport and/or chargetransport. While the term phosphorylation is used in the specific sense,the embodiments described herein are intended to cover otherattachment/detachment of protein cofactors. In an exemplary embodimentof the invention, the electric field is applied directly or induced, forexample, using induction coils, magnetic fields or by methods of chargetransport in the tissue, such as changing of ionic concentration.

Some embodiments of the invention are based on the surprising discoveryby the inventors that an electric field can have an immediate effect onphosphorylation of proteins.

In some embodiments of the invention, modification of protein expressionand/or mRNA expression are practiced, in addition to or instead ofphosphorylation changes. In an exemplary embodiment of the invention,protein levels of at least two proteins are normalized by application ofan electric field.

In an exemplary embodiment of the invention, the modification method isused as a therapy and/or in diagnosis, for example for a diseased heartand/or for other organs in the body.

In an exemplary embodiment of the invention, the modification of theprotein activity is a relatively direct result of the method, ratherthan a side effect. This directness can be noticed, for example in thetime scale of some embodiments of the invention, where a change inphosphorylation is noticeable within a few seconds or minutes.Alternatively or additionally, the directness can be noticed in a lackof intermediates, for example as indicated by the change inphosphorylation taking place even in cell homogenate, or even in thepresence of protein inhibitors.

In an exemplary embodiment of the invention, protein activitymodification comprises changing the actual work done by proteins in thecell. Optionally, as noted herein, this work is modified withoutsynthesis of new proteins, for example, by modification ofphosphorylation and/or activation of pathways.

In an exemplary embodiment of the invention, the modification is evidentwithout requiring the synthesis of new protein. For example,phosphorylation of existing proteins may be provided.

In an exemplary embodiment of the invention, modification of proteinactivity and/or phosphorylation comprises modifying a steady stateaverage level of such phosphorylation. Optionally, this modificationcomprises periodically increasing the percentage of phosphorylatedproteins. Alternatively or additionally, the modification is achieved byshifting a balance between phosphorylation and dephosphorylation. Insome embodiments, the modification relates to a single heart beat.

In an exemplary embodiment of the invention, the modification is on thetime scale of a single or small number of heart beats, or faster (e.g.,1 second or faster). Optionally, an effect of the modification isnoticeable on a same time scale.

In an exemplary embodiment of the invention, the affected proteins areproteins that act on a non-protein substrate, for example calciumchannel proteins. Alternatively or additionally, the proteins aresignaling proteins and/or act on other proteins and/or are acted on byother proteins. For example, the affected protein enhances the activityof various enzymes, which, in turn, regulate essential ion channels andpumps. One example is that phospholamban, when phosphorylated, canincrease the activity of calcium ATPase also known as SERCA-2a.

An aspect of some embodiments of the invention relates to usingphosphorylation as a target for therapy, especially a therapy where thetherapy method can be controlled at a relatively fast rate. For example,phosphorylation of phospholamban leads to increased activity of calciumATPase and/or increase of the affinity of calcium ATPase pump forcalcium. This, in turn, leads to increased or enhanced uptake of calciumfrom the cell cytosol to the sarcoplasmic reticulum (SR).

In an exemplary embodiment of the invention, changing phosphorylationwill stabilize the cell biochemistry and prevent or undo decline in cellfunctionality. This may allow a general improvement in the patient tooccur, for example, as natural feedback and healing processes can kickinto action.

In an exemplary embodiment of the invention, an indication of proteinactivity other than phosphorylation is used for example, either asfeedback or to determine efficacy, for example, microbiologicalparameters such as SR calcium level or macro parameters such as cardiacoutput as a function of MVO₂.

In an exemplary embodiment of the invention, the target of therapy isachieving a relative phosphorylation level. Alternatively oradditionally, the target is achieving an absolute phosphorylation level.Such a target may be a window, with the values of the window optionallybeing variable. In one example, the values are dependent on one or moreof 1) the state of phosphorylation of the protein, 2) the availabilityof the protein itself, 3) the condition and viability of the organ, 4)stressful conditions imposed upon the organ as a result of dailyactivity and/or 5) resulting from variation in circadian rhythm.

In an exemplary embodiment of the invention, the target of therapy isachieving an effect of change in phosphorylation, for example, a changeof cellular function, caused for example, by rebalancing a previouslyupset balance between the activity of various proteins or setting a newset point of a new, less hazardous balance (e.g., for therapy of acondition where the balance is hazardous).

In an exemplary embodiment of the invention, the target is achieving acertain value or profile of protein activity, for example, a certaincalcium pumping rate or pumping profile (as dependent on concentrationand/or cell stress), which value or profile is determined by thephosphorylated protein, alone or in conjunction with other associatedproteins or cellular mechanisms.

In an exemplary embodiment of the invention, when the phosphorylation isused as a target, the therapy is increased, decreased and/or otherwisemodified based on its actual or expected effect on phosphorylation. Inan exemplary embodiment of the invention, feedback is provided for themethod by measuring phosphorylation directly or indirectly.

In an exemplary embodiment of the invention, phosphorylation is used asa negative or minimum target. For example, when it is not possible toachieve a desired effect on an entire heart, for example, due to powerlimits or side effect limits, a minimum phosphorylation target is setfor various parts of the heart and the therapy is configured so thatthis minimum achievable target is achieved. In another example, it maybe desirable to minimize the amount of phosphorylation, for example inthe event that hyperphosphorylation leads to progressive worsening ofdisease. In this case, the pulse sequences applied may be optimized tominimize the global and/or regional expected or measured phosphorylationof a particular protein. In an exemplary embodiment of the invention,phosphorylation modification in ischemic and non-ischemic regions isdifferent or otherwise dependent on the condition of underlying tissue.For example, ischemic regions being controlled to improvephosphorylation and non-ischemic regions are controlled to also increasecontractility.

In an exemplary embodiment of the invention, phosphorylation is used asa single or as one of several parameters in optimization or selection ofa pulse sequence.

In an exemplary embodiment of the invention, phosphorylation is used asa guide for electrification sequence parameters settings. In some casesit may not be practical to use phosphorylation as an indicator forfeedback, however, general experiments (such as those described herein)may show that certain sequences have a certain effect onphosphorylation. In an exemplary embodiment of the invention, sequencesthat are defined for another effect, for example, contractilitymodulation, are modified to take into account the results of theseexperiments. In another example, a sequence that is shown to have acertain phosphorylation effect is used in an open or semi-open loop,rather than a closed loop. The phrase “semi-open loop” is used to meanthat feedback and modification of the sequence is at a much slower ratethan the application of the sequence, for example, once a week or once amonth for a sequence applied once an hour or more often.

An aspect of some embodiments of the invention relates to kits andmethods of use thereof. In an exemplary embodiment of the invention, akit includes a means to analyze a tissue sample to determine someindication of its phosphorylation levels, protein expression levels,mRNA levels and/or other biochemical markers. Optionally, the kitincludes 1, 4, 10, 20, 25 or more or intermediate numbers of biochemicalmarker detectors for markers as described herein and/or additionalmarkers.

In an exemplary embodiment of the invention, the type and/or severity ofthe disease is classified using the expression results and/or responseto treatment results for a plurality of genes/proteins, for example, 3,5, 10, 20 or more. In an exemplary embodiment of the invention, adatabase is built up by storing typical results for different patientsand detecting relationships between levels/responses that are associatedwith certain outcomes and/or pathologies. It is expected that withincrease in the number of biochemical markers and/or treatments thatsuch classifications can be detected.

In an exemplary embodiment of the invention, the kit includesinstructions for use, optionally including thresholds of expected valuesand/or changes in values expected over time. Optionally, the kit isuseful for one or more of diagnosis, detecting treatment progress and/orclassifying patients according to expected responsiveness.

In an exemplary embodiment of the invention, the kit is included withsuitable software which tracks values and/or provides results.

In an exemplary embodiment of the invention, the kit is used whiledevice implanting, to assess a suitable implantation area of electrodesin the heart for example, according to the response to acute stimulationindicated by the kit.

Optionally, the kit is used by taking a tissue biopsy, for example,using a needle or a catheter and testing the levels of bio-chemicals ina biopsy sample.

In an exemplary embodiment of the invention, the kit is used for activetesting of a tissue sample. A tissue sample is extracted and optionallyhomogenized (e.g., using a separate device) and then an electric fieldor other treatment is applied to the sample. Depending on the responseof the sample, a diagnosis, progress and/or classification isdetermined. Optionally, the kit is provided with a set of electrodeswhich can be selectively attached to an implantable device, to assessits effect on homogenate. Alternatively or additionally, a stand-aloneelectrification system is used. Optionally, this stand-alone systemincludes a controller adapted to apply multiple electrification schemesand optionally store the effect of each scheme. Optionally, thestand-alone device includes a sampling chamber for selecting a part ofthe tested sample and applying a test thereto, for example, determininginstantaneous or near instantaneous phosphorylation.

In another example, the kit is used to test pre-treatment levels ofbiochemicals, including, for example, phosphorylation level.

In an exemplary embodiment of the invention, the kit is packaged as aseparate entity, while in some cases, multiple kits may be used, whichmay be packaged as a set. Optionally, different kits for different setsof biochemical markers are provided. Alternatively or additionally, thekits are provided, for example, as single or multiple one-time kits,with a cardiac controller, to be used as part of the implantation and/orelectrode location searching process.

Optionally, the kit is used during implantation, before implantation isfinalized, to help decide if the device should be left in the body ornot, depending on its acute efficacy, for example, on phosphorylation.

Optionally, a kit is used to test treatment other than electricaltreatments, for example, drug treatments or exercise treatments.Optionally, the kit is used by sampling a sample before and after thetreatment or by applying a proposed treatment to the sample itself andseeing if the sample exhibits a positive effect.

An aspect of some embodiments of the invention relates to controllingtissue, for example soft tissue and/or non-cartilagous tissue and/ornon-supporting tissue such as the heart, by directly affecting a balancepoint of phosphorylation or other biochemical activities therein.

In an exemplary embodiment of the invention, the electrical activity ofa cell is considered as having a resetting effect on the cell. Applyinga field at the time of activation of the cell may miss this resetperiod. In an exemplary embodiment of the invention, a field whichmodifies the cell balance is applied before/after resetting time, then,when the resetting is applied by cellular activation, the cell is at anew balance point. In some cases, multiple {set balance; reset} cyclesare applied to achieve a desired change in a cell or population ofcells. In some cases, rest periods between applications are required,for example, to allow a cell to stabilize and/or find a new balancestate absent external effects.

In an exemplary embodiment of the invention, an electric field isapplied which skews a balance between phosphorylation anddephosphorylation of a protein. This skewing optionally includes a longterm increase in phosphorylation, for example if the time constants ofdephosphorylation are higher than those of dephosphorylation. In anexemplary embodiment of the invention, the protein affected isphospholamban or an ion channel, for example a trans-membrane calciumchannel.

In an exemplary embodiment of the invention, the effect is a short termeffect, for example, by applying the field in a manner which allows longterm phosphorylation levels to recover and thus prevent a long termchange in cellular behavior, while providing an acute effect. In aparticular example, the field is applied for a short time and thenstopped until (e.g., according to measurement or estimation) thephosphorylation levels received. Optionally, the field is applied for ashort enough time that the total acute change is small, for example afew percent (e.g., <10 percent). Optionally, a mix or intermediatesituation between short and long term effect is provided. Optionally,both large acute changes and gradual long term changes are provided, forexample with long term changes being on the range of hours and acutechanges seconds or minutes.

Optionally, the field is applied often enough to cause a long termeffect. Optionally, the frequency of application causes only a slowchange in acute values, optionally causing no acute effects to be seen.

In an exemplary embodiment of the invention, the applied field ismodified to take into account the change in cellular behavior and/orchange in phosphorylation.

It is noted that a test field applied for testing tissue response maynot be the same as the treatment field. In one example, the test fieldis stronger. In another example, the treatment field is modified basedon the results of the test field.

In an exemplary embodiment of the invention, the balance betweenphosphorylation and dephosphorylation is tipped to restore a correctbalance. Alternatively or additionally, the balance is skewed to beabnormal, for example to drive a cellular homeostasis mechanism in adirection to correct a defect in cellular behavior and/or compensate forsuch behavior.

Optionally, the applied electric field is a dual function field, forexample, being used for pacing or preventing arrhythmia. Optionally, theapplied field does not acutely (e.g., within 10 heart beats or fewer)increase contractility by more than 3%, or less.

A broad aspect of some embodiments of the invention relates tonon-immediate effects of therapy. A first type of non-immediate effectis an effect that lasts a considerable amount of time after theapplication of the therapy. This type of effect may allow relativelylong non-therapy periods between therapy application times, while stillproviding useful treatment of a patient. A second type of non-immediateeffect is an effect that lasts after therapy is stopped, for example,physical and/or biochemical remodeling of the heart or cells thereof. Athird type of non-immediate effect is an effect that only becomesnoticeable after a time, for example, protein expression changes whichare not associated with immediate (acute) hemodynamic changes.

An aspect of some embodiments of the invention relate to new therapeuticnon-excitatory sequences for the heart. Optionally, these sequences haveas an aim to improve phosphorylation, rather than only contractility andin some cases, without immediate improvement in contractility.Optionally, a phosphorylation improving sequence, while generallycapable of contractility enhancement, is applied at too low a repetitionrate and/or power to achieve a meaningful change in contractility. Anexample is calsequestrin that, when phosphorylated, increases thesequestration of calcium into the sarcoplasmic reticulum but does notincrease contractility.

In an exemplary embodiment of the invention, the sequences are optimizedto one or more of acute or longer term effects of one or more ofphosphorylation, protein and/or mRNA levels.

Acute effects have some potential benefits for use as feedback,including one or more of faster feedback so faster optimization and/orper patient optimization can more easily be achieved, relativesteadiness of physiological condition during optimization and/or abilityto control an ongoing process, such as titrating of therapeutic drugsparticularly in i.v. type drugs or delivery of any drug and dose.

In an exemplary embodiment of the invention, the optimization (includinga semi-optimization) is on a per patient, per tissue (e.g., location inheart), per diagnosis and/or per patient classification group.

In an exemplary embodiment of the invention, as compared tocontractility modifying signals, the sequences have a lower duty cycleand/or more quiet periods between sequences, designed such that adesired phosphorylation effect is achieved, even if a sufficient chargeis not delivered each beat (or any beat) to cause significant increasein contractility. In an exemplary embodiment of the invention, thesequence is based on a delivery of minimum signals that increasephosphorylation, at time intervals timed so that decay ofphosphorylation between applications is smaller than or the same as theincrease achieved by an application. Optionally, the delay betweensignals and/or signal length and/or other parameters vary over time totrack a possibly varying effect of the signal on phosphorylation asphosphorylation changes.

In some cases, a field which would otherwise reduce contractility (e.g.,a hyperpolarizing field) is used.

In an exemplary embodiment of the invention, a power saving sequence isdefined, which, for example, is designed to maintain phosphorylationlevels, even if a desired contractility enhancement is not directlyachieved, by reducing pulse amplitude, frequency of application and/orother power-related parameters. In some cases, contractility is not aconsequence of the phosphorylation normalization.

In an exemplary embodiment of the invention, a minimum dosage sequenceis defined, which achieves a desired phosphorylation effect, withoutnecessarily achieving other immediate beneficial effects such ascontractility enhancement effects. Long-term, the improvement inphosphorylation may also improve contractility. In an exemplaryembodiment of the invention, a therapeutically effective sequencecomprises applying a field to the heart less often than once in 5minutes, once in 10 minutes, once in 30 minutes, once an hour, once aday and/or once a week. For some uses, a set of signals, for example,10, 20 or 30 signals (each signal corresponding to one heart beat), maybe applied at such intervals, with an optional inter-signal spacing.

In an exemplary embodiment of the invention, a phosphorylation-effectingsignal comprises applying signals at different times in the cardiaccycle, such as absolute or relative refractory periods and excitatoryperiod. The signal may be synchronized to the heart as a whole or tolocal activity, for example. Optionally, the signal is excitatory insome times of application. Optionally, the signal, at some embodimentsthereof, may be applied at any point in the cycle, or at least during60%, 80% or more of the cycle.

In an exemplary embodiment of the invention, the optimizing of the pulsesequence is based on selecting a pulse or pulse parameters which willhave a desired effect on the patient, for example, phosphorylation,e.g., above 10% increase, 20%, 40%, 100%, 200%, 500%, 1000% orintermediate or larger percentage increases. In some cases, a decreaseis desired, for example, a decrease of 20%, 40%, 70%, 90% orintermediate or greater percentage reductions. Not all such increasesand/or decreases are available for all biochemicals.

In an exemplary embodiment of the invention, a method of manufacturingis provided in which a pacemaker or another electrical field applyingdevice is programmed to have a pulse known to have a desired biochemicaleffect, such as phosphorylation, optionally even if such pulse has areduction in other effect.

An aspect of some embodiments of the invention relates to applyingtherapy, for example, electro-biochemical control therapy as describedherein, by selecting a long term therapy effect (e.g., as describedherein) and modifying the therapy to match the effect. In an exemplaryembodiment of the invention, the modifying comprises changing the dailyduration of signals, modifying rest periods between signals and/orchanging the applied signals. Optionally, the modifications are betweenpatients and/or within a patient, for example, as therapy progresses.Optionally, secondary targets for optimization when modifying a therapyare total applied charge and existence of side effects (positive ornegative). Other parameters as described herein may be varied as well.

In an exemplary embodiment of the invention, one or more of thefollowing targets is selected: ejection fraction elevation. Cardiacmuscle dimensions (e.g., reduction), chamber volume (e.g., reduction),quality of life (e.g., as measured using various tests), peak O2consumption (e.g., increase), anaerobic threshold (e.g., improve), 6 mwalking distance, fluid retention, sleep apnea severity and/or episodesand/or exercise tolerance.

In an exemplary embodiment of the invention, the patients selected fortherapy are those not indicated for cardiac resynchronization therapy.

In an exemplary embodiment of the invention, patients selected have anormal QRS (not wide, no conduction problems) and/or no desynchrony.

In an exemplary embodiment of the invention, patients have a narrow QRS.

Optionally, the patients are NYHFA class III-IV severity patients.

An aspect of some embodiments of the invention relates to controlling aheart taking into account differences between local and remote effectsof a treatment such as electrical field application.

In an exemplary embodiment of the invention, a local area is an areawhich is directly affected by the treatment, for example a tissue arealying between two electrodes that are electrified or an areas to which apharmaceutical is provided, for example using a path or using localinjection or using other methods known in the art. In an exemplaryembodiment of the invention, the tissue in this area is used to detectimmediate effects of the field, for example, change in phosphorylationand changes in contractility. Optionally, a sensor is provided at thelocal area for example, a sensor that measures local muscle functionand/or biochemical behavior, which sensor generates an indication of theeffect of the sequence. Optionally, a one time use sensor is used, forexample an anti-body covered optical fiber. Optionally, several suchsensors are provided.

Alternatively or additionally to acute measurements within minutes orseconds, measurements on a scale of hours are made.

In an exemplary embodiment of the invention, the remote area is in thesame heart chamber or in a different heart chamber and serves toindicate general progress in the cardiac condition. Optionally, suchgeneral progress is detected by measuring changes in biochemical markersin such remote tissue.

Optionally, a treatment aims to improve one or both of local and remoteeffects.

In an exemplary embodiment of the invention, areas to treat are selectedbased on a desired local and/or remote effect. In one example, local(e.g., electrode application) areas are selected such that a generalimprovement in cardiac function and a subsequent remote effect may beexpected. In another example, multiple local areas are selected so as topositively control the cellular behavior in those areas, for examplesimultaneously or in series.

In an exemplary embodiment of the invention, progress is measured bydetecting a wave-like propagation of tissue improvement, starting at thesites of electrode application. Such sites may be selected to provide adesired such propagation of improvement over the heart. Alternatively oradditionally, progress is detected by measuring gradual improvement inmultiple locations simultaneously. Optionally, if improvement ismeasured using biopsies, different locations are sampled each time.

In an exemplary embodiment of the invention, electrode location areselected so as to best utilize exciting tissue resources, for example,enhance weak tissue rather than strong tissue or optimize use of bloodflow resources. Optionally, the treatment areas are selected to increaseblood demand and drive angiogenesis. Optionally, treatment is applied atareas where blood flow is reduced, as some treatments do not increaseoxygen demand.

In an exemplary embodiment of the invention, electrode placement isselected to provide a desired stretching behavior to nearby tissue.Alternatively or additionally, electrode placement is selected tominimize diffusion or travel distances for biochemicals between treatedareas and other areas.

In an exemplary embodiment of the invention, a local area is 20 cm² 10cm², 5 cm², 3 cm², 2 cm², 1 cm² or greater or smaller or intermediatesizes.

An aspect of some embodiments of the invention relates to applying aphosphorylation affecting signal on generally non-contracting tissue,such as plugs, transplants and/or scar tissue (especially at boundariesthereof). In an exemplary embodiment of the invention, this applicationis used to stabilize and/or improve phosphorylation levels in suchtissue. In an exemplary embodiment of the invention, tissue plugs areremoved and treated and then reinserted back into the heart (autograft).Optionally, the grafts are inserted into scar tissue.

In an exemplary embodiment of the invention, plugs are extracted wholefrom tissue. Alternatively, plugs are built up and treated before,during and/or after build-up. In one example, plugs are formed bysettling tissue on a matrix.

In an exemplary embodiment of the invention, apparatus is provided forholding a plurality of tissue plugs (e.g., 3, 5, 10, 20 or more) whilean electric field is applied thereto, for example, the apparatusincluding a chamber with physiological fluid, the chamber optionallyincluding supports for the plugs. Optionally, one or more plugs aresampled or tested to see an effect of eth field. Optionally, one or moreelectrodes are provide din or adjacent the walls of said chamber.

In an exemplary embodiment of the invention, stimulation of scar tissuecan cause it to regain mechanical activity, for example by stimulationand/or healing of dormant tissue therein.

In an exemplary embodiment of the invention, stimulation of a transplantis used to enhance its activity and/or prevent degradation due toremoval and implant. Optionally, the stimulation used does not causesignificant mechanical activity, for example, being applied at long timeintervals. Optionally, the signal is applied to cooled, non-contractingtissue. Possibly, phosphorylation of a particular protein can lead toactivity that stimulates the release, for example of specificneurohormones and activation of essential proteins. Optionally, theapplication of the signal to a cooled or cardioplegic heart is usedduring cardiac and/or brain surgery to facilitate restarting of theheart after such surgery.

An aspect of some embodiments of the invention relates to detecting ofchanges in biochemical behavior in the heart.

In an exemplary embodiment of the invention, changes in ECG morphologywhich indicate changes in protein levels and/or phosphorylation, aredetected. Optionally, the morphology is a single cell clamp measurement.

In an exemplary embodiment of the invention, a catheter biopsy is usedto extract tissue.

In an exemplary embodiment of the invention, a tissue sample isextracted and tested by stimulation/treatment thereof outside the body.Optionally, the tissue is homogenized and/or separated into individualcells.

In an exemplary embodiment of the invention, biochemical state isdetermined by measuring reactivity to other biochemicals. For example,the responsiveness to beta blockers may be detected to change whencertain proteins are phosphorylated.

Optionally, antibody based tracers are used, for example, in conjunctionwith florescent dyes and/or radioactive materials.

In an exemplary embodiment of the invention, biochemical changes aredetected by identifying macroscopic properties of the heart. In oneexample, changed protein expression is expected to increase conductionvelocity or maximum contraction velocity. Optionally, these parametersare measured and used to detect and/or estimate a change due to proteinexpression. Optionally, a database calibrating changes in macroscopicparameters with biochemical parameters, is provided. In an exemplaryembodiment of the invention, conduction velocity is measured orestimated from measured ECG signals, for example, measured using thedevice. Optionally, these changes are measured at a time that a fieldapplication device is not active, for example, not active for a periodof a few minutes, hours or days.

A broad aspect of some embodiments of the invention relates to complextherapy utilizing modification of tissue behavior. In one exemplaryembodiment, electrical tissue biochemical behavior modification is usedtogether with pharmaceutical provision to achieve a synergistic effect,for example, one therapy compensating for failing of the other therapyor two therapies acting to achieve a common goal. One example isbeta-blocker therapy in which an initial reduction in cardiac output maybe offset using an electrical therapy. After beta blocker therapy has apositive effect, electrical therapy may be used to provide a furtherincrease in cardiac improvement. Optionally, a same pathway or mechanismis targeted using multiple therapies. In another exemplary embodiment,multiple pathways are treated, some with biochemical behaviormodification using electrical means and some, optionally, with othermeans, such as pharmaceuticals. In another example, another therapy orapplication is used to modify the effect of electrical therapy, forexample, applying or reducing stress before or during electrical therapyapplication. It should be noted that in some cases contractility changeis minimal, absent or in the form of reduction.

An aspect of some embodiments of the invention relates to targetedtherapy delivery and/or modulation of therapy. In an exemplaryembodiment of the invention, a signal that modulates phosphorylation isapplied, while the availability of a substrate relevant forphosphorylation is modified. In one example, a pharmaceutical whichreduces or increases the phosphorylated protein is provided. In anotherexample, the electric field is used to activate proteins generated usinggene therapy, such as DNA plasmid injection coding for SERCA-2a, wherebyphosphorylation of phospholamban would enhance the activity of theSERCA-2a. In an exemplary embodiment of the invention, targeting isachieved by therapy requiring the temporal and spatial intersection ofthe substrate/precursor and the signal which has the phosphorylationeffect. Optionally, an area is drained of or filled with substrate, forexample, by previous application of suitable signals, exercises and/orpharmaceuticals. For example, a cardiac region may be stressed toincrease or reduce its susceptibility to the phosphorylation modifyingsignal.

In an exemplary embodiment of the invention, it is noted that the needof a substrate to be available for a protein to be phosphorylated allowsselective achievement of the contractility modulation effect and thephosphorylation effect, for example by selectively applying the signalwhen there is no substrate and/or by selectively applying the signalsoften enough to achieve phosphorylation but not often enough forsignificant contractility enhancement.

In an exemplary embodiment of the invention, particular proteins areselectively affected by timing the lengths of signals applied so thatthey differentially affect one protein or another. Optionally, thesignals are repeated in order to have a sufficient effect on a desiredprotein. Optionally, the signals are delayed from one another in orderto allow changes in activity levels of a protein to decay. Optionally,selective mRNA expression is provided by selectively affecting proteinswhich cause mRNA changes.

In an exemplary embodiment of the invention, selective inhibitors areprovided, for example, anti-sense DNA or protein inhibitors, to inhibitcertain biochemical pathways. Alternatively or additionally, substratesor exciting materials are provided to enhance certain pathways.

An aspect of some embodiments of the invention relates to selectivecontrol of different proteins, for example selective phosphorylationrates thereof. In an exemplary embodiment of the invention, an electricfield is used to differentially affect more that one of phospholambanand calcium channels. Such differentiability is to be expected due tothe difference in location in the cell of the two proteins(trans-membrane and intracellular) and due to the different mechanismfor dephosphorylation, each of which generally has a different timerate. Thus, applying pulses of electricity at a certain amplitude and/ora certain rate may be expected to affect one protein more than theother. Phosphorylation and dephosphorylation rates are optionallycontrolled by controlling availability of substrates and/or catalyticenzyme, for example, using suitable bio-chemicals applied to the cell orpatient.

It should be noted that multiple mechanisms for improving contractilitygenerally exist in a cell. Each of the calcium channels and thephospholamban, affect contractility in a different manner and this mayallow selecting what manner of affect is desired.

In an exemplary embodiment of the invention, contractility increase of acell is blocked using one biochemical, while using phosphorylationcontrol to improve cellular homeostasis. Optionally, such blocking isapplied locally, for example to small parts of the heart where theoverall cardiac output will not be too damaged. Optionally,anti-arrhythmic treatment (e.g., electrical or drug) is applied at asame time.

In an exemplary embodiment of the invention, a local effect is appliedto enhance local function of the heart, for example in viable regions ofa ventricle after massive myocardial infarction. Alternatively oradditionally, a local effect is applied to suppress the over contractionof a region of the heart as in patients with hyperdynamic septum as inasymmetrical septal hypertrophy.

In an exemplary embodiment of the invention, tissue viability (e.g.,after infarct, donor organ) is tested using methods as described hereinfor examining activity.

In an exemplary embodiment of the invention, one or more of thefollowing mechanism are addressed using electrical therapy as describedherein: affecting calcium availability (intra- or inter-beat timescale), affecting phosphorylation (intra- or inter-beat time scale,affecting mRNA expression directly or indirectly (e.g., by calciumavailability and/or phosphorylation) and protein synthesis (days) whichaffects the cellular steady state. By suitable mixing of therapies orgiving therapies with opposite effects at suitable timing, variousresults can be achieved, for example, long term improvement due toprotein synthesis may be offset by momentary blocking of proteinactivity (e.g., with a pharmaceutical). Similarly, once proteinsynthesis is underway, a delay of a few days will not completely undothe effects of therapy. In a particular example, prior to starting withbeta blockers, electrical therapy may be applied for a while and then,once synthesis has started, e.g., after a few days or a week or based onmeasurements, electrical therapy may be stopped or applied only everyfew days, while beta-blockers (or other pharmaceuticals) are applied.

There is therefore provided in accordance with an exemplary embodimentof the invention, a method of modifying tissue behavior, comprising:

determining a desired modification of tissue behavior for at least oneof treatment of a disease, short or long term modification of tissuebehavior, assessing tissue state and assessing tissue response tostimulation;

selecting an electric field having an expected effect of modifyingprotein activity of at least one protein as an immediate response of atissue to the field, said expected effect correlated with said desiredmodification; and

applying said field to said tissue.

In an exemplary embodiment of the invention, said applying has a localeffect only on said tissue.

In an exemplary embodiment of the invention, said tissue comprisescardiac tissue.

In an exemplary embodiment of the invention, at least one of said atleast one protein is an SR protein.

In an exemplary embodiment of the invention, at least one of said atleast one protein is not sensitive to physiologically occurringinter-cellular electric fields.

In an exemplary embodiment of the invention, at least one of said atleast one protein is not an ion transport protein.

In an exemplary embodiment of the invention, at least one of said atleast one protein controls another protein.

In an exemplary embodiment of the invention, said at least one proteincomprises phospholamban.

In an exemplary embodiment of the invention, said at least one proteincomprises a trans-membrane calcium channel.

In an exemplary embodiment of the invention, said at least one proteincomprises a plurality of proteins. Optionally, said plurality ofproteins belong to at least 2 separate biochemical control pathways.Alternatively or additionally, said plurality of proteins belong to atleast 3 separate biochemical control pathways. Alternatively oradditionally, said plurality of proteins belong to at least 4 separatebiochemical control pathways. Alternatively or additionally, saidseparate pathways are protein interaction pathways. Alternatively oradditionally, said separate pathways include genomic control.

In an exemplary embodiment of the invention, modifying protein activitycomprises attaching or detaching a cofactor to at least on of said atleast one protein.

In an exemplary embodiment of the invention, modifying comprisesphosphorylation.

In an exemplary embodiment of the invention, modifying comprisesdephosphorylation.

In an exemplary embodiment of the invention, modifying protein activitycomprises modifying the activities of existing proteins, withoutsynthesizing new proteins.

In an exemplary embodiment of the invention, said immediate responsecomprises a response within less than 10 minutes. Alternatively oradditionally, said immediate response comprises a response within lessthan 2 minutes. Alternatively or additionally, said immediate responsecomprises a response within less than 20 seconds. Alternatively oradditionally, said immediate response comprises a response within lessthan 2 seconds. Alternatively or additionally, said immediate responsecomprises a response within less than 0.5 seconds.

In an exemplary embodiment of the invention, said modifying is atransient modification temporally correlated with said applying.

In an exemplary embodiment of the invention, said modifying is apersistent modification lasting at least 10 times the length of saidapplying. Optionally, said modifying is a persistent modificationlasting at least 100 times the length of said applying.

In an exemplary embodiment of the invention, said modifying comprisesmodifying a ratio between protein configurations of different activationlevels of at least one of said at least one protein by a factor of atleast 1.2. Optionally, said factor is at least 2. Alternatively oradditionally, said factor is at least 5.

In an exemplary embodiment of the invention, determining a desiredmodification comprises determining a desired modification of tissuebehavior. Optionally, said modification is a short term modificationsignificant within 3 hours. Alternatively or additionally, saidmodification is a long term modification significant within 3 weeks.Alternatively or additionally, said modification is a long termmodification which comprises changes in protein expression levels.Alternatively or additionally, said change is a change in at least 5proteins associated with said behavior. Alternatively or additionally,said change does not include a change in expression of at least twohousekeeping genes.

In an exemplary embodiment of the invention, determining a desiredmodification of tissue behavior comprises determining said modificationfor treating a disease. Optionally, treating comprises increasingcontractility. Alternatively or additionally, treating comprisesreversing a heart failure state in said tissue. Alternatively oradditionally, said reversing comprises reversing on a cellular level.Alternatively or additionally, treating comprises normalizing proteinexpression levels. Alternatively or additionally, treating comprisesnormalizing protein activity levels. Alternatively or additionally,treating comprises skewing protein activity levels to compensate forsaid disease. Alternatively or additionally, treating comprises changingcellular homeostasis to a different set point. Alternatively oradditionally, treating comprises modifying said treatment using amodification of protein activation levels as a target of said treating.

In an exemplary embodiment of the invention, modifying compriseschanging a balance between activation and deactivation of a protein insaid tissue.

In an exemplary embodiment of the invention, determining a desiredmodification of tissue behavior comprises determining said modificationfor assessing of tissue state. Optionally, said assessing comprisesassessing based on said tissue response to said applying. Alternativelyor additionally, said assessing comprises assessing based on a responseof said tissue to said applying. Alternatively or additionally, saidassessing comprises assessing based on tissue biochemical markers.Alternatively or additionally, assessing comprises classifying at leastone of a disease state and disease severity. Alternatively oradditionally, assessing comprises selecting a treatment according tosaid tissue response. Alternatively or additionally, assessing comprisesassessing during an implantation procedure for a therapeutic device.Alternatively or additionally, assessing comprises assessing during aset-up stage for a therapeutic device. Alternatively or additionally,assessing comprises assessing as part of an on-going therapy using atherapeutic device. Alternatively or additionally, assessing comprisessampling said tissue for analysis thereof. Alternatively oradditionally, assessing comprises selecting a placement for at least oneelectrode based on said assessing.

In an exemplary embodiment of the invention, said tissue comprises atissue sample.

In an exemplary embodiment of the invention, said tissue comprisesin-vivo tissue.

In an exemplary embodiment of the invention, said tissue comprisesseparated cells.

In an exemplary embodiment of the invention, said tissue comprisesbroken down tissue in which cells are broken down.

In an exemplary embodiment of the invention, said tissue comprisestissue homogenate.

In an exemplary embodiment of the invention, said determining a desiredmodification of tissue behavior comprises determining a modification forassessing a tissue response to stimulation.

In an exemplary embodiment of the invention, the method comprisesmodifying a selected field according to a response of said tissue tosaid applying. Optionally, said modifying a selected field comprisesimproving said field with respect to a desired effect of said field onsaid tissue.

In an exemplary embodiment of the invention, the method comprisesprogramming a therapeutic device with said improved field.

In an exemplary embodiment of the invention, the method comprisesmeasuring an immediate response of said tissue to said field.

In an exemplary embodiment of the invention, the method comprisesmeasuring a non-immediate response of said tissue to said field.

In an exemplary embodiment of the invention, the method comprisesmeasuring a non-local effect on remote tissue physiologically associatedwith said tissue in response to said field.

In an exemplary embodiment of the invention, said field isnon-excitatory for said tissue.

In an exemplary embodiment of the invention, said tissue is contractiletissue and wherein said field reduces contraction of said tissue.

In an exemplary embodiment of the invention, applying comprises applyingsaid field in conjunction with a pharmaceutical, which has aninteraction with an effect of said field on said tissue.

In an exemplary embodiment of the invention, modifying protein activitycomprises modifying protein activation levels.

In an exemplary embodiment of the invention, applying said field is byinduction.

There is also provided in accordance with an exemplary embodiment of theinvention, apparatus for treating tissue, comprising:

at least one electrode adapted to apply an electric field to in-vivotissue;

a controller including a memory having stored therein at least oneelectric field sequence which modifies protein activity levels in saidtissue, said controller being configured to determine that amodification of said protein activity is desired and apply said sequencein response said determination. Optionally, said controller memory hasstored therein a plurality of sequences or sequence parameters andwherein said controller is configured to select between the sequences orparameters. Alternatively or additionally, the apparatus comprises aninput and wherein said controller makes said determination according toa signal received on said input.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of manufacturing a therapeutic device comprising:

selecting a pulse sequence according to its effect on protein activitymodification; and

programming a controller of said therapeutic device to apply saidsequence.

In an exemplary embodiment of the invention, said sequence iselectrical.

In an exemplary embodiment of the invention, said sequence is selectedto treat heart failure.

There is also provided in accordance with an exemplary embodiment of theinvention, a therapeutic device manufactured by the methods describedherein.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of tissue treatment, comprising:

providing a plurality of tissue plugs;

applying an electric field to said plugs to modify biochemical behaviorthereof; and

implanting said plugs. Optionally, said plugs are cardiac tissue plugs.

In an exemplary embodiment of the invention, the method comprisesexcising tissue of said plugs from a same heart into which the plugs arelater implanted.

In an exemplary embodiment of the invention, the method comprisesgenetically modifying said plugs prior to said implantation.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of therapy, comprising:

selectively applying a therapy material to a tissue; and

selectively modifying protein activation in said tissue utilizing asecond therapy. Optionally, said therapy material is gene therapymaterial and wherein selectively modifying comprises selectivelymodifying protein activity of a protein generated as a result of saidtherapy.

In an exemplary embodiment of the invention, said therapy material is asubstrate for a protein and wherein selectively modifying comprisesselectively modifying protein activity of said protein.

In an exemplary embodiment of the invention, selectively applyingcomprises making said substrate inaccessible to said protein.

In an exemplary embodiment of the invention, said therapy materialincreases the availability of a protein and wherein selectivelymodifying comprises selectively modifying protein activity of saidprotein.

In an exemplary embodiment of the invention, said second therapycomprises applying an electric field.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of modifying tissue behavior, comprising:

determining a desired modification of tissue behavior for at least oneof treatment of a disease, short or long term modification of tissuebehavior, assessing tissue state and assessing tissue response tostimulation;

selecting a tissue modifying activity having an expected effect ofmodifying protein activation levels of at least one protein as animmediate response of a tissue to the activity, said expected effectcorrelated with said desired modification; and

applying said activity to said tissue.

In an exemplary embodiment of the invention, said activity comprises apharmaceutical.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of modifying tissue behavior, comprising:

selecting a desired balance between a pair of agonist and antagonistreactions in a cell; and

applying an electric field to said cell such that said field modifies anexisting balance towards said desired balance. Optionally, said balanceis a balance between phosphorylation and dephosphorylation.

There is also provided in accordance with an exemplary embodiment of theinvention, a biochemical assaying kit, comprising:

an indicator of protein phosphorylation; and

instructions for using said phosphorylation as an indicator of tissuestate. Optionally, said instructions comprise software.

In an exemplary embodiment of the invention, said kit includes at leastone electrode adapted to apply an electric field to a sample beingtested with said kit.

In an exemplary embodiment of the invention, the kit includes a chamberand including a sampler adapted to remove a sample for assaying.

In an exemplary embodiment of the invention, the kit is adapted for usewith a controller adapted to affect tissue in the body using an electricfield.

In an exemplary embodiment of the invention, the kit comprises aplurality of indicators for a plurality of protein or mRNA expressionlevels.

There is also provided in accordance with an exemplary embodiment of theinvention, apparatus for treating cardiac dysfunction, comprising:

at least one electrode adapted to apply an electric field to tissue of apatient; and

a controller configured to apply an electrical sequence in spurts ofapplications with delays between the spurts, said field being configuredto have an affirmative modifying effect which modifies a behavior ofsaid tissue in a positive manner, such that a lasting effect from aspurt continues for a significant time after the spurt. Optionally, saidlasting effect has a wash-out period.

In an exemplary embodiment of the invention, a total effect of saidcontroller is to modify protein expression levels in a heart of saidpatient.

In an exemplary embodiment of the invention, said lasting effectcomprises enhanced tissue function of tissue to which said field isapplied.

In an exemplary embodiment of the invention, said lasting effectcomprises enhanced tissue function of tissue to which said field is notapplied.

In an exemplary embodiment of the invention, said field is anon-excitatory field.

In an exemplary embodiment of the invention, said delay is at least 1minute.

In an exemplary embodiment of the invention, said delay is at least 5minutes.

In an exemplary embodiment of the invention, said delay is at least 10minutes.

In an exemplary embodiment of the invention, said spurt is applied forless than a single heartbeat.

In an exemplary embodiment of the invention, said spurt is applied forless than 3 seconds.

In an exemplary embodiment of the invention, said spurt is applied forless than 10 seconds.

In an exemplary embodiment of the invention, said spurt is applied forless than 100 seconds.

In an exemplary embodiment of the invention, said field increasescontractility.

In an exemplary embodiment of the invention, said controller is adaptedto measure washout response to a spurt for said patient.

In an exemplary embodiment of the invention, said delay is at least 3times a length of said spurt.

In an exemplary embodiment of the invention, said delay is at least 10times a length of said spurt.

In an exemplary embodiment of the invention, said delay is at least 50times a length of said spurt.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of treating a patient with an electrical therapy,comprising:

applying an electrical field to an organ of the patient;

stopping said application for a length of time which is a function of anexpected washout time of an effect of said field.

In an exemplary embodiment of the invention, said organ is a heart andwherein said electric field enhances cardiac function.

In an exemplary embodiment of the invention, said organ is a heart andwherein said electric field enhances cardiac output on a level of asingle heartbeat.

In an exemplary embodiment of the invention, said effect is an immediateeffect. Alternatively or additionally, said effect is a short-termeffect. Alternatively or additionally, said effect is a long-termeffect.

In an exemplary embodiment of the invention, the method comprisesrepeating said applying and said stopping at least 20 times. Optionally,said stopping time varies between repetitions. Alternatively oradditionally, said stopping time is varied as the number of hours ofapplication during a day.

In an exemplary embodiment of the invention, said effect is selected tobe a change in one or more of ejection fraction, cardiac muscledimension, cardiac chamber dimension, quality of life as measured by aquestionnaire, peak oxygen consumption, anaerobic tolerance, 6 meterwalk distance, fluid retention, sleep apnea severity, and exercisetolerance.

In an exemplary embodiment of the invention, the method comprisesselecting a patient for therapy responsive to the patient not having anincreased QRS length or desynchrony.

In an exemplary embodiment of the invention, said therapy isnon-excitatory.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of therapy location placement for therapy of tissue,comprising:

applying a test therapy to the tissue; and

deciding on suitability of the placement based on an effect of proteinactivity levels of said test therapy. Optionally, said test therapy isapplied outside the body.

In an exemplary embodiment of the invention, said therapy is electricaltherapy for the heart.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of therapy location placement for therapy of tissue,comprising:

providing an organ to be treated; and

selecting at least one location of treatment, according to a desiredpropagation of biochemical effect of said treatment in said organ.Optionally, said propagation is a mechanical propagation. Alternativelyor additionally, said propagation is a biochemical propagation.

In an exemplary embodiment of the invention, said at least one locationcomprises a plurality of locations.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of therapy location placement for therapy of tissue,comprising:

applying a test therapy to the tissue; and

deciding on suitability of the placement based on an effect of proteinactivity levels of said test therapy, even if an improvement in organfunction is not detected.

In an exemplary embodiment of the invention, said test is applied to apart of an organ separate from the organ.

In an exemplary embodiment of the invention, said therapy is electricaltherapy.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of therapy, comprising:

applying a therapy at a first location;

determining if the therapy is having a first effect by measuring a shortterm response at said first location; and

determining if the therapy is having a second effect by measuring along-term response at a second, untreated, location. Optionally, themethod comprises tracking progression of said therapy based onimprovement of said second location.

In an exemplary embodiment of the invention, said therapy is electricaltherapy.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of treating cardiac tissue, comprising:

selecting a tissue with reduced oxygen transport thereto; and

applying an electric field to said tissue, which field does not reduceactivity thereof. Optionally, said field increases contractility of saidtissue. Alternatively or additionally, said field reduces oxygenconsumption of said tissue.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of assessing tissue state, comprising determiningbiochemical activity, concurrently in relation to biochemical markersassociated with at least two genes associated with heart failure.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of assessing cardiac tissue state, comprisingdetermining biochemical activity, concurrently in relation tobiochemical markers associated with at least two genes.

In an exemplary embodiment of the invention, the method comprisesassessing tissue state in response to a therapy applied thereto.

In an exemplary embodiment of the invention, said assessing is inresponse to at least 5 markers concurrently. Alternatively oradditionally, said assessing is in response to at least 10 markersconcurrently. Alternatively or additionally, said assessing is inresponse to at least 20 markers concurrently. Alternatively oradditionally, said markers include mRNA expression levels. Alternativelyor additionally, said markers include protein expression levels.Alternatively or additionally, said markers include protein activitylevels.

In an exemplary embodiment of the invention, the method comprisesimproving a therapy using said biochemical markers as a target.

In an exemplary embodiment of the invention, said biochemical markersinclude GATA-4.

In an exemplary embodiment of the invention, said biochemical markersinclude phosphorylation of phospholamban.

In an exemplary embodiment of the invention, said determining comprisesdetermining an immediate effect.

In an exemplary embodiment of the invention, said determining comprisesdetermining a short term effect.

In an exemplary embodiment of the invention, said biochemical markersinclude markers from at least two pathways in the tissue.

There is also provided in accordance with an exemplary embodiment of theinvention, a kit adapted to perform the determining as described herein.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of treating cardiac dysfunction, comprising:

determining a desired effect on protein activity; and

applying a field to cardiac tissue to cause such desired change.

In an exemplary embodiment of the invention, said desired effectcomprises a selective effect on fewer than 5 proteins.

In an exemplary embodiment of the invention, said desired effectcomprises a selective effect on fewer than 10 proteins.

In an exemplary embodiment of the invention, said desired effectcomprises a selective effect on fewer than 40 proteins.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of treating cardiac dysfunction, comprising applyingan electric field to said heart which is sufficient to have asignificant normalization effect on protein phosphorylation levelswithout significant effect on contractility.

There is also provided in accordance with an exemplary embodiment of theinvention, apparatus for delivering an electric field to cardiac tissue,being programmed to use a minimum amount of power sufficient to affectpositively the phosphorylation of HF-related proteins.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of treating tissue, comprising:

determining a desired effect on levels of at least one of phospholambanand phosphorylation thereof; and

applying a field to tissue to cause such desired change.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of treating cardiac tissue, comprising:

selecting a signal according to its not being expected to increase meanoxygen consumption; and

applying an electric field with said signal to cardiac tissue, whichfield does not reduce activity thereof.

There is also provided in accordance with an exemplary embodiment of theinvention, a method of manufacturing a cardiac device, comprising:

selecting a signal which is expected not to increase mean oxygenconsumption while not reducing tissue activity; and

programming a cardiac device with said signal.

There is also provided in accordance with an exemplary embodiment of theinvention, a cardiac controller, comprising:

a signal application circuit adapted to apply a first signal whichincreases mean oxygen consumption and second signal which does notincrease said oxygen consumption; and

a control circuit adapted to control said signal application circuit toselectively apply one of said two signals, based on oxygen availability.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the invention will be described withreference to the following description of exemplary embodiments, inconjunction with the figures. The figures are generally not shown toscale and any sizes are only meant to be exemplary and not necessarilylimiting. In the figures, identical structures, elements or parts thatappear in more than one figure are preferably labeled with a same orsimilar number in all the figures in which they appear, in which:

FIG. 1 is a schematic diagram of a tissue controller utilizing anelectrical field to achieve a phosphorylation effect, in accordance withan exemplary embodiment of the invention;

FIGS. 2A-2O show the effect of a short term or chronic (multi-monthapplication period) CCM signal on proteins and mRNA in the heart, inaccordance with an exemplary embodiment of the invention;

FIGS. 3A-3E show the immediate effect of CCM signals on proteins inaccordance with exemplary embodiments of the invention;

FIG. 4 is a flowchart of a method of therapy taking phosphorylation intoaccount, in accordance with an exemplary embodiment of the invention;

FIGS. 5A-5R are blots showing relative protein levels (in dogs) forcontrol, heart failure and heart failure with chronic CCM application,in accordance with an exemplary embodiment of the invention;

FIG. 6 shows mRNA expression levels in a cardiac septum (where signalsare applied), in a chronic study, in accordance with an exemplaryembodiment of the invention;

FIGS. 7A and 7B show a general lack of acute effect of CCM on tissueremote from a CCM delivery site, in accordance with an exemplaryembodiment of the invention;

FIG. 8 shows levels of phosphorylated phospholamban in dog septum withheart failure with chronic treatment, in accordance with an exemplaryembodiment of the invention;

FIG. 9A and FIG. 9B show rise times and decay times for treatment usinga CCM signal, in accordance with an exemplary embodiment of theinvention;

FIG. 10 presents western blot data illustrating effect of CCM accordingto an exemplary embodiment of the invention on protein levels of CSQ,SERCA-2a, PLB and RyR in the LV free wall and inter-ventricular septum;

FIG. 11 presents western blot data illustrating effect of 3 months ofCCM according to an exemplary embodiment of the invention on levels ofP-PLB at serine-16 and threonine-17 in the LV free wall andinter-ventricular septum relative to sham-operated HF dogs and normaldogs;

FIGS. 12A and 12B present western blot data and bar graphs illustratingeffect of CCM according to an exemplary embodiment of the invention onthe ratio of P-PLB to total PLB relative to untreated HF dogs in the LVanterior wall at the site of signal delivery (FIG. 12A), and in the LVposterior wall remote from the site of CCM signal delivery (FIG. 12B);

FIGS. 13A and 13B present mRNA (FIG. 13A) and protein blots (FIG. 13B)illustrating expression of Sorcin in LV tissue of HF Dogs treated withCCM for 3 months according to an exemplary embodiment of the invention;

FIGS. 14A, 14B, 14C and 14D present mRNA (FIGS. 14A and 14C) and proteinblots (FIGS. 14B and 14D) of the Presenilin-1 (FIGS. 14A and 14B) andPresenilin-2 (FIGS. 14C and 14D) in LV tissue of HF Dogs treated withCCM for 3 months according to an exemplary embodiment of the invention;and

FIGS. 15A and 15B present mRNA (FIG. 15A) and protein blots (FIG. 15B)illustrating expression of Calstabin in LV tissue of HF Dogs treatedwith CCM for 3 months according to an exemplary embodiment of theinvention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Overview

Exemplary embodiments of the present invention are based on thediscovery that certain electrical signals have an immediate effect onthe phosphorylation of certain cardiac proteins, for example, at leastone, at least 2, at least 3 or at least 5 proteins. Optionally, a set ofproteins is affected, for example proteins that relate to calciumavailability. In particular, an effect on proteins that control calciumpumping and others (all of which are known to change in heart failure),has been discovered. Selectively in which proteins were affected is alsoshown. Proteins not related to heart failure are apparently notaffected. The effect has been found to work in a range of tissueorganization levels starting from tissue homogenate, through isolatedcells and even in in-vivo dog hearts. The effect remains (in dog hearts)for 15 minutes at least, and an initial effect is noticeable after aslittle as 1 minute or even a few seconds, such as 3-10 seconds, fortissue samples from a foci of signal application. Some proteins werephosphorylated and some were un-phosphorylated by the signal. Generally,the results show a change in the phosphorylation values in a directionof “normal” levels, or their being maintained at normal values.Experiments using pacing signals have not shown this effect.

This phosphorylation effect is optionally in addition to or instead ofeffects on mRNA expression and protein expression levels, which, in someembodiments of the invention are normalized, changed to more normalvalues and/or changed in a manner which overcompensates for a deficit.

In accordance with exemplary embodiments of the invention, thisdiscovery is used in the design of methods and apparatus for treatingtissue, especially cardiac tissue.

For purposes of this specification and the accompanying claims, theterms “expression” and “regulation” should be construed in theirbroadest possible sense so that they include any factor which influencesa biological activity. Thus expression and/or regulation include, butare not limited to factors such as control exercised at the genomic DNAlevel, the RNA level, the protein level and secretion level.

With regard to genomic DNA, control may be exercised, for example, byaltering a phosphorylation and/or a methylation state at one or moresites in a relevant genomic sequence.

With regard to RNA, control may be exercised, for example, by regulatinga rate of transcription of an mRNA transcript, and/or by altering astability of an mRNA transcript, and/or by altering a relative amount ofsplice variants from a single mRNA transcript.

With regard to protein, control may be exercised, for example, byregulating one or more cleavage events and/or addition of one or moreside chains and/or protein stability. In some cases cleavage of aprotein may increase a biological activity of the protein and/orfacilitate secretion from a cell. In other cases, cleavage of a proteinmay reduce or eliminate activity of the protein. The term side chains,as used herein, denotes any molecular moiety which can be attached to anamino acid of the protein. In some cases, side chains are attached aftertranslation. In other cases a tRNA molecule may attach an amino acidbearing a side chain during translation.

With regard to secretion, control may be exercised, for example, byallowing or preventing of secretion of compounds, such as connectivetissue dissolving enzymes and biochemical signaling molecules.

Schematic Device

FIG. 1 is a schematic diagram of a tissue controller 100 utilizing anelectrical field to achieve a phosphorylation effect, in accordance withan exemplary embodiment of the invention. In an exemplary embodiment ofthe invention, controller 100 comprises at least one electrode 102adapted to apply an electric field to a tissue 104, for example a heart.A control circuitry 106 optionally controls the output of a power drive108 which electrifies the at least one electrode 102. One or moreoptional physiological sensors 110 optionally provide feedback to beused by circuitry 106 in controlling the electrification of the at leastone electrode 102, for example, general feedback about the heart (e.g.,ECG) and/or a micro-biological sensor, for example for mRNA expressionprofiles or protein activity level. Optionally, such sensors includefiber-optic anti-body based sensors, DNA or protein chips and/orlab-on-chip type sensors. General body sensors, such as a sensor 112 maybe used as well, for example, to estimate fluid retention or motion ofthe patient (e.g., rest/exercise as estimated using an accelerator).

In an exemplary embodiment of the invention, controller 100 is in theform of a pacemaker or non-excitatory controller, for example asdescribed in one or more of the following applications and publications.

Exemplary protocols for the actual delivery of signals to the heartand/or implantation of wires to deliver CCM signals is set forth in PCTpublication No. WO 97/25098 and U.S. Pat. No. 6,317,631, both of whichare incorporated herein by reference in their entirety. Following is alist of patents and publications which describe apparatus and methodswhich may be useful in conjunction with the present invention, thedisclosures of all of which are incorporated herein by reference, as arethe disclosures of all publications mentioned in this application:

Cardiac output enhanced pacemaker, U.S. Pat. No. 6,463,324, ApparatusAnd Method For Controlling The Contractility Of Muscles, U.S. Pat. No.6,233,484, Controlling Heart Performance Using A Non-Excitatory ElectricField, U.S. Pat. No. 6,317,631, Muscle Contraction Assist Device, U.S.Pat. No. 6,285,906, Modulation Of Intracellular Calcium ConcentrationUsing Non-Excitatory Electrical Signals Applied To The Tissue, PCTWO01/24871 and PCT WO00/12525, Electrical Muscle Controller, U.S. Pat.No. 6,363,279, Electrical Muscle Controller using a Non-ExcitatoryField, U.S. Pat. No. 6,330,476, Cardiac Output Controller, U.S. Pat. No.6,298,268, Cardiac Output Enhanced Pacemaker, U.S. Pat. No. 6,463,324,Sensor Based Regulation of Excitable Tissue Control of the Heart,WO00/27475, Regulation of Excitable Tissue Control of the Heart based onPhysiological Input, WO00/27476, Trigger Based Regulation of ExcitableTissue Control of the Heart, U.S. Pat. No. 6,587,721, Pacing withHemodynamic Enhancement, PCT WO/1998/023211A1, ETC Delivery via RVSeptum, PCT WO0182771A3, Anti-Arrhythmia Device having CardiacContractility Modulation Capabilities, PCT WO01/30445, andAnti-Arrhythmic Device & a Method for Delivering Anti-Arrhythmic CardiacTherapy, PCT WO01/30139.

In some embodiments of the invention, the devices described in PCTpublications WO 2005/056111, WO 2005/102188 and/or WO 2005/105013, withsuitable changes in programming, are used.

In an exemplary embodiment of the invention, controller 100 includes amemory in which various measured and/or expected values and behaviors oftissue and bio-chemicals are stored. Controller 100 may be implanted,only the electrodes implanted or be wholly outside the body, optionallyonly with a sensor implanted, optionally with a sensor inserted asneeded.

In an exemplary embodiment of the invention, controller 100 includes amolecule source 114 or controls an external molecule source (not shown),for example, using a data line or a wireless communication. Optionally,the molecule source sends a signal to controller 100 that a molecule wasprovided. Alternatively, an external trigger is used for both controller100 and molecule source 114. Optionally, the source is provided using atube or other delivery system 116. Such a source may be used, forexample, as described below with reference to FIG. 4.

In an exemplary embodiment of the invention, controller 100 uses one ormore external sensors 120 (e.g., body ECG) and/or an external control118 to guide its behavior. Optionally, an external programmer/monitor isused for communication from a patient to a caregiver, for example, ahospital or monitoring agency. Optionally, such a programmer/monitor isused to send programs and/or setting to controller 100 and/or sendqueries regarding the effects of certain treatment sequences.Optionally, controller 100 maintains a log of physiological measuresand/or applied sequences and/or synchronization thereof. The log may belimited to times before and after the treatment and/or include randomtimes or times triggered by various physiological measurements.

Controller 100 optionally provides one or more of the therapeuticeffects listed following this paragraph. Optionally, when two or moreeffects are provided, the effects are provided simultaneously oralternately (e.g., alternating or otherwise intermixed electrificationsignals to have the multiple desired effects). In some cases, thebehavior of the controller is modified to provide a tradeoff betweenmultiple effects. Some of the effects may be enhanced and/or caused bymodification of protein activity and/or mRNA expression. In some cases,the sequences used for “standard” effects are modified so that desiredbiochemical effects as described herein are achieved. Such modificationmay be by per patient optimization or by off-line generalquasi-optimization independent of any particular target patient.Optionally, the controller is programmable, for example, using outsideprogramming to perform one or more of the following therapies, asdescribed elsewhere:

a) pacing;

b) contractility enhancement;

c) cardiac resynchronization;

d) conduction velocity modification;

e) remodeling;

f) arrhythmia treatment and/or prevention;

g) healing of diseased cardiac tissue; and

h) stabilizing cardiac condition.

Phosphorylation Modification

As noted above and as will be described below, it has been discoveredthat pulses originally designed for cardiac contractility modification(CCM) have an immediate and/or a long range effect on genomic expression(e.g., as evidenced by mRNA expression) and/or on protein activity.However, it is believed that other pulses also have such effects. Inparticular, CCM pulses often require a certain timing, which may not berequired for some phosphorylation effects. In particular also, CCMpulses may need to be applied more often for significant CCM effectsthan for significant phosphorylation effects. In particular also,phosphorylation effects may be achieved even when CCM or at leastcontractility increase is reversed or null or fluctuates. In particularalso, phosphorylation effects may also be achieved using excitatorysignals or a combination of excitatory and non-excitatory signals andnot only with pure non-excitatory signals.

In an exemplary embodiment of the invention, within seconds of applyingthe electric field to the tissue in-vivo or in-vitro, existingphospholamban protein is phosphorylated, without the need to synthesizemore protein, but rather making use of what is already there in a“dephosphorylated state”. In some embodiments, while the phosphorylationprovides an “immediate response”, the treatment and/or phosphorylationact as a trigger for later synthesis or change in synthesis of proteins.

In an exemplary embodiment of the invention, one or both of between beatand within beat phosphorylation levels are controlled. For example, toreduce inter-beat effects, phosphorylation increase is kept at a levelwhich cellular homeostasis mechanism can counteract by the next time afield is applied. In some cases, this requires one or both of control offield amplitude and frequency of application.

Optionally, different proteins are differently controlled, for exampleintra-cellular and trans-membrane proteins.

FIG. 4 is a flowchart of a method of therapy taking phosphorylation (orother biochemical change) into account, in accordance with an exemplaryembodiment of the invention.

At 402 a diagnosis of a patient is optionally made. Optionally, thepatient is re-diagnosed as his therapy advances.

At 404, one or more proteins to be affected by the electrical signalsare selected. Optionally, the proteins are selected based on thediagnosis. Optionally, a set of related proteins, for example, calciumavailability proteins, are selected. Optionally, one or more locationsin the heart to be affected are selected. Optionally, target protein,mRNA and/or phosphorylation levels and/or other tissue or organ effectsare selected.

At 406, additional considerations are selected and/or rejected. In oneexample, the additional consideration is that the heart be paced and/orits LVEF (left ventricular ejection fraction) be increased. In adifferent example, considering that the pulses may be applied less oftenthan pacing pulses, more painful treatments, such as external pulses,are used. Optionally, pro-arrhythmic pulses are used, for example, ifthe treatment is under a hospital setting or if the treatment isinfrequent enough so that the total danger of arrhythmia over a timeperiod is within acceptable parameters. Controller 100 optionallyincludes a defibrillation and/or fencing function applied through thesame or other electrodes. In an exemplary embodiment of the invention,immediate reduction in cardiac efficiency is acceptable, as this isunder controlled conditions and increased cardiac health will followshortly (e.g., within minutes, hours, days or weeks).

In another example, a protein modifying signal is applied as an excitingsignal. In another example, a protein modifying signal is applied inparts of the cardiac cycle where it reduces contractility (or dP/dt)and/or prevents normal signal propagation in the heart. Optionally, thepart of the heart to which the signal is applied is decoupled ordeactivated, for example, by cold, by fencing or by various cardioplegiadrugs.

In an exemplary embodiment of the invention, it is assumed that theprotein modification signal has an effect that is at least partlyindependent of the point in the cardiac cycle at which it is applied, atleast for some proteins, such as proteins that are not electricallysensitive to the electrical cycle in the cell. For example, the effectdepends on the availability of ATP for phosphorylation and not on theparticular charge conditions in the cell. For some applied signals, theion concentrations may have an effect on the efficacy of the signal andthese may be dependent on the phase in celldepolarization/repolarization cycle. In an exemplary embodiment of theinvention, the effect of a particular sequence and/or for a particularpatient is taken into account when deciding on the strength and/or otherparameter of a signal.

In an exemplary embodiment of the invention, it is assumed that aCMM-type signal has multiple effects on cardiac tissue which may betargeted separately, at least to some extent. Some effects arecausative, but may be at least partially decoupled or require multipleinputs to generate a particular desired result. Some of the effects are:

a) Effect on tissue polarization. This may include, for example,hyper-polarization, pacing and depolarization.

b) Effect on repolarization/depolarization cycle. This may include, forexample, extending a plateau duration.

c) Effect on tissue function (external), for example, increasedcontraction strength and inhibition of mechanical and/or electricalactivity.

d) Effect on protein phosphorylation.

e) Effect on genomic expression.

f) Short vs. long term effects (e.g., remodeling).

At 408 a pulse sequence and/or application schedule (e.g., once a week)are optionally generated or selected, for example, using a look-up tableor by searching for a solution. Many optimization and search methods areknown in the art and may be used to help determine a treatment protocol;for example, linear programming, hill climbing and trial and errorexperimentation (e.g., manual or automatic experiments). The particularcharacteristics of the tissue and/or patient may also be determined, forexample, by experimentation or by a table linking disease type to anexpected (and/or desired) effect of a change in the protocol. Thegeneration is optionally performed on controller 100. Optionally, thegeneration is at a treatment center, for example, if the patient comesin periodically for treatment or, if treatment is by remote means, byusing a telephone link to control an implanted or external field source.

At 410, the sequence is applied to the tissue.

At 412, compensation for the effects of the sequence may be provided, ifnecessary, for example, anti-arrhythmia treatment or oxygen provision.Optionally, the compensation is provided before and/or during thesequence application, or intermingled therewith.

At 414 an additional therapy and/or cofactor are optionally provided,which optionally interact synergistically with the sequence, forexample, on a cellular level or by one assisting the other to have aneffect. In one example, the additional therapy is pharmaceutical. Inanother example, the additional therapy provides a cofactor or substratewhich the proteins need to change their activity level. In anotherexample, DNA therapy is made more specific by the proteins beinggenerated and/or being activated by the field. In another example,exercise or rest is enforced so as to build-up a supply of substrate(e.g., protein or phosphor) on which the field can have an effect.

In an exemplary embodiment of the invention, the additional therapy isapplied systemically. Alternatively or additionally, at least oneadditional therapy is applied locally, for example, using targetingmethods and/or local delivery methods as known in the art. Optionally,the methods of PCT publications WO 01/93951, WO 00/74773 and/or WO01/93950, the disclosures of which are incorporated by reference, areused. Optionally, in accordance with some of these methods, a sameelectrical field source applies both a therapeutic-effect field and atargeting/transport field.

At 416, the effect of the field is optionally measured. Optionally, themeasurement is in substantial real-time. In an exemplary embodiment ofthe invention, a gene or protein chip are used to detect protein,phosphorylation and/or mRNA levels. Alternatively or additionally, anoptical sensor is used, for example an anti-body carrying opticaldetector. Optionally, the sensor is consumable and lasts, for example,for 5, 10, 20 or 100 uses (e.g., a multiplicity of single use sensorsmay be supplied). Optionally, spectroscopy methods are used, forexample, Raman spectroscopy.

While phosphorylation may be measured directly, optionally, cellularand/or organ behavior characteristics are measured instead, for example,stroke volume and effect on ECG.

In an exemplary embodiment of the invention, evaluation methods asdescribed in U.S. provisional application Ser. No. 60/765,974, filedFeb. 7, 2006, by inventors Benny ROUSSO et al., the disclosure of whichis incorporated herein by reference, are used.

In an exemplary embodiment of the invention, evaluation is used toevaluate one or more of patients state, disease state and/or diseaseprogression. Evaluation means may be included in controller 100 or beexternal to patient. Evaluation may be applied relatively continuously,for example more often than once a day, or less often for example,weekly or monthly or less often. In an exemplary embodiment of theinvention, evaluation is used to identify events where changes intherapy are desirable, for example, where therapy was insufficient,where patient reached his bounds or where adverse effects are found.

In an exemplary embodiment of the invention, evaluation comprisesdetecting changes in cardiac activation due to change sin conductionvelocity. Optionally, the changes are detected by detecting a change inrelative timing of events on an impedance measurement of the heart.

At 418, the sequence is optionally repeated, optionally being modifiedaccording to the obtained results.

Optionally, multiple feedback loops are maintained, for example, someparameters being measured on a second by second or minute by minutebasis and others being measured on an hourly, daily, weekly and/ormonthly schedule.

Optionally, the measurements are off-line, for example, by biopsytaking. Optionally, the sample is frozen, for example in liquidnitrogen, to prevent changes. The results are optionally transmitted tocontroller 100.

In an exemplary embodiment of the invention, the intended effect of theelectrical therapy is to tip a balance between phosphorylation anddephosphorylation mechanisms in the cell. For example, the electricfield can be applied so that a protein (such as calcium channel) is moreeasily phosphorylated, while dephosphorylation mechanisms stay the same(or vice-versa). This can cause both an immediate (intra-beat) effect onphosphorylation levels and, possibly depending on the ratio between theimmediate effect and the dephosphorylation mechanism, can cause a longerterm increase.

In some cases, the long term increase is carried past normal levels, forexample, to force a certain operation state of the controlled tissue.

In an exemplary embodiment of the invention, the electrical modificationof proteins is used to achieve an effect that does not directlytranslate into long term changes in protein levels.

In an exemplary embodiment of the invention, the electrical modificationis used to trigger a change in cellular state. For example, once certaincellular balances are upset, cellular mechanism will then change theoperational mode of the cell in a desired manner.

In an exemplary embodiment of the invention, the electrical modificationis used to support a failing cellular mechanism so that the cell canrecover.

In an exemplary embodiment of the invention, electrical modification ofbiochemical behavior works together with existing control mechanism ofthe body, in addition to or instead of over-expression orunder-expression of body biochemicals. In an exemplary embodiment of theinvention, existing control mechanism are not disrupted, but ratherutilized, for example, using existing control mechanism to controlfunctionality provided by the therapy, causing existing controlmechanism to act when they are inhibited from acting for some reasonand/or inhibiting over-reactive control mechanism.

In an exemplary embodiment of the invention, the electrical modificationis used to damp or overcome an over-protective or run-awayprotection/control mechanism. One example, in cardiac cells, is amechanism that when the cell feels over stressed, reduces contractilityso that the cell can funnel its resources to viability. The electricalmodification can be used to suppress this mechanism, so thatcontractility can resume, especially if the cell is actually capable ofcontraction and such contraction is suppressed or reduced by a run-awaymechanism. Optionally, if there is a degradation in function, forexample as detected by reduction in cardiac output or degraded ECGsignals, the protein modification is stopped and is used as anindication that the cellular protection mechanism was not actually beingover protective.

In an exemplary embodiment of the invention, the electrical modificationincreases calcium availability, thereby allowing existing controlmechanism to “decide” if this increased availability should be utilizedat any given instant to increase cardiac output. It is noted that theavailable increase and ability to work with existing mechanism canprevent degradation of heart tissue.

In an exemplary embodiment of the invention, the therapy does notrequire any particular ligand or effector to tie to, but acts directlyon bio-molecules.

In an exemplary embodiment of the invention, the electrical modificationis not treated as a systemic therapy, but as a local therapy, forexample, limited in effect to tissue directly or indirectly affected.

Exemplary Considerations for Pulse and/or Schedule Design

Further to the examples above, following are exemplary considerations tobe taken into account during sequence and/or schedule design. Inparticular, pulse length, power, shape, intra-pulse delay, repetitionrate and/or delay between sequences may be modified and/or optimized tohave a desired effect (or mainly such a desired effect).

a) Pulse rate and length. Protein specificity is optionally achievedbased on one or both of length of each pulse and delay between pulses.As noted, some proteins are significantly affected by short pulses. Sucha protein can be selectively affected by using shorter pulses thanneeded for other purposes, and repeating the pulses at inter-pulsedelays shorter than a relaxation time for the affected protein.Optionally, proteins are targeted based on their location in the celland the type (e.g., amplitude, frequency and/or waveform) of pulse thatis expected to penetrate sufficiently into the cell and/or affect theparticular protein. Optionally, pulse rate and/or delays and/or lengthare modified as needed to achieve a target. This allows for targetingthe effect to a limited number of proteins.

b) mRNA vs. protein effects. In an exemplary embodiment of theinvention, mRNA and/or protein effects are selected by applying pulseswhich have a short term effect on proteins but the effect is not longenough to trigger significant mRNA expression effects. For example, if aprotein phosphorylation level is not elevated for long enough, it may bethat mRNA effects will be absent. However, phosphorylation may beincreased multiple discrete times (with delays between them). Inaccordance with some embodiments of the invention, some mRNA effects aredirectly determined by proteins, so that protein levels may becontrolled in order to achieve selective mRNA effects.

c) Counter effects. In an exemplary embodiment of the invention, thecontrol is selected so that mRNA effects and phosphorylation effects arenot synchronized. For example, long plateaus of increasedphosphorylation may be used to increase mRNA effect, but totalphosphorylation modification may be selected to be insignificant over atime period. One reason for this may be lack of sufficient blood flow tothe heart, so that acute changes are less desirable than gradualchanges. In another example, pharmaceuticals which counteract an effectmay be provided to effectively select certain effect(s). It is notedhowever, that phosphorylation changes associated with increasedcontractility did not show increased oxygen demand, in some experimentsat least and in at least one experiment, even reduction in oxygendemands.

d) Stability. Optionally, the long term effect of treatment is a newbalanced set point between the various proteins. However, in the shortterm, such a balancing need not be achieved. Optionally, the heart iscontrolled so that the various proteins are not at a balance with regardto their respective activities.

e) Physiological time scales. In an exemplary embodiment of theinvention, the rate of application and/or duration relates to the timescales of physiological behavior, for example, sleep/wake cycles, eatingtimes, time scale for formation of protein and/or mRNA transcription(e.g., 1-3 days and 1-4 hours, respectively).

f) Triggering. In an exemplary embodiment of the invention, a pulsesequence is applied and/or modified in response to a trigger event. Inan exemplary embodiment of the invention, controller 100 has storedthereon (or on an external controller) a table of situations andresponse (e.g., different sequence application parameters).Alternatively or additionally, to a table, other programming means maybe used, for example, logic and neural networks. Exemplary triggerevents include, stress, arrhythmia, ischemia, eating, resting, changesin concentration of biochemicals (e.g., intra-cellular, extra-cellularand/or blood) and/or changes in electrical signals (e.g., ECG, EMGand/or EEG). Possibly, this allows the sequences to be applied onlywhere there is a need and/or when they will have a significant effect.

g) Treatment of areas and combination of therapies. In an exemplaryembodiment of the invention, treatment is applied to multiple areas.Optionally, sequences are not applied in parallel to different areas,but in series. For example, one area being treated and then a secondarea is treated. Optionally, the duration of each treatment and/or cycletime depend on one or more of stabilization times, relaxation timesand/or length of effect times. In one example, each area is treated onehour on and several hours off, with other areas (e.g., 2, 3, 4) beingtreated in the “off” time. In an example of therapy combination, if theallowed delay between applications of one therapy are longer than theapplication times of another therapy, the two therapies can be appliedin a mixed manner to tissue. In some cases, two therapies can be appliedsimultaneously, for example, signals with different electricalfrequencies, each of which has a selective effect on differentbiochemical pathways, for example, one signal which affects nerves(e.g., high frequency) and a second signal which affectsphosphorylation.

One example is found in skeletal muscles, where their biochemical state(aerobic or anaerobic) can depend on nerve stimulation. Optionally,nerve stimulation is used to prompt cells to be more (or less) sensitiveto phosphorylation control of phospholamban.

h) Noticeability of effects. In some cases, it may be desirable toprevent any immediate effects (e.g., contractility changes), while stillproviding therapy. For example, if only 10% of beats are treated, thismay not be noticeable on a daily basis, but the cumulative effects ofprotein synthesis will be noticeable after a while. Optionally, thesequence is provided in a manner which will not be noticeable over timescales of minutes (e.g. 1-20), hours (e.g., 1-10) and/or days (e.g.,1-7). In an exemplary embodiment of the invention, the lack ofnoticeability is used to prevent interfering with the patient'slifestyle.

i) Compliance. In an exemplary embodiment of the invention, electricalfield application does not require compliance from the patient and thiscan be a benefit. Optionally, applying the signal at a time when thepatient is asleep, can prevent the patient from causing (intentionallyor unintentionally) physiological states where the therapy is notapplied for various reasons (e.g., safety, suitability). Optionally,however, compliance is required, for example, for taking of a medicationthat works together with the therapy. Optionally, if a patient does notindicate to the system that the medication was taken (e.g., using awireless communication device), or if this lack is sensed by the system,the applied sequence is changed, initiated and/or inhibited, tocompensate for this lack.

j) Adaptation. In general, electrical signals are expected to be lesssusceptible to adaptation than pharmaceuticals, because the duration ofelectrical therapy and its onset can be controlled better than forpharmaceuticals. In an exemplary embodiment of the invention, providingthe therapy in a manner which is specific in time, prevent adaptationmechanism that relate to the mere existence of the therapy. Optionally,the therapy is changed over time, to prevent and/or counteractadaptation. Optionally, the therapy is changed when an adaptation issuggested by lack of or reduced reaction of the patient.

In general, short term effects/goals and long term effects/goals may beat odds.

Exemplary Pulse Properties

While CCM pulses as described herein may be used, optionally, the pulsesused are modified, for example, to save power and/or reduce the need forsynchronization.

In an exemplary embodiment of the invention, the applied pulses and/orsequences require considerably less power than CCM signals (e.g., 7.73volts for 33 ms each 45 seconds), for example, only 20%, only 10%, only5%, only 1%, or intermediate or smaller power usage. Optionally, thepower per pulse is maintained, but the number of pulses in a time periodis reduced, so that a cumulative power level is reduced (e.g., ascompared to CCM signals).

In an exemplary embodiment of the invention, the amplitude and/orduration used is insufficient for contractility, for example, beingunder the amount (if any) which causes a 20%, 10%, 3%, 2%, 1% orintermediate or smaller increase in contractility over a period of 5minutes from initial application. For example, the application rate,power and/or duration are smaller.

In an exemplary embodiment of the invention, the voltage used is lowerthan for CCM, for example, being 0.1, 0.5-1 volts, or less, or valuessuch as 2V or 3V or other values smaller than 8 Volts. It should benoted that in the results shown below, the CCM signal was clearly morethan required to achieve a meaningful phosphorylation, and thus a signalless powerful may be suitable. Larger voltages such as 10, 20 or 30volts may be used in some embodiments.

In an exemplary embodiment of the invention, the duration of the pulsesis as short as 1 ms (with an optional associated increase in power), orlonger, such as 10 ms, 20 ms or more. Alternatively, the signal may belengthened, for example, being 50, 100, 150, 200, 300, 400 ms or more.Optionally, medication which increases a refractory period is used inconjunction with long pulses. Optionally, fast and short term actingmedication is used during pulse application.

In an exemplary embodiment of the invention, a total charge carried by aphosphorylation pulse is at least 5, 10, 30, 50 or 100 times the chargecarried by a pacing pulse, such as a 3V 0.75 ms pulse.

High power pulses are optionally applied as sub-threshold (forexcitation) pulses.

In an exemplary embodiment of the invention, the current for the pulseis between 0.2 ma and 20 mA, or intermediate or higher values. Otherexemplary values for current (maximum current) are 0.4, 0.8, 1, 3, 7 or10 mA (or intermediate values).

In an exemplary embodiment of the invention, the applied signalcomprises a series of pulses, for example, each pulse being bi-phasic,with each phase being 5.5 msec (˜100 Hz), applied in synchronizationwith a local pacing activity (e.g., at a delay thereto). Optionally, theseries is of 2-3 pulses, or a larger number, for example, 5, 10, 20 ormore or intermediate numbers.

Other waveforms can be used, for example, sinus waves or triangularwaves. Optionally, a delay is provided between pulses of a series.Optionally, a pulse includes both excitatory and non-excitatorycomponents.

In an exemplary embodiment of the invention, signals applied outside theabsolute refractory period are applied at lower amplitudes. The relevantthresholds are optionally determined by experimentation or usingstandard values (noting that diseased tissue may have a lower thresholdand/or abnormal refractory periods. Optionally, medication is providedto extend the refractory period and allow a greater charge and/or longerpulse sequence to be delivered during a single beat.

In an exemplary embodiment of the invention, a tune-up of the pulseparameter is carried out, for example, to enable power to be reduced toa minimum which has an effect and/or as the patient response changes.

In an exemplary embodiment of the invention, the application scheduleincludes reducing the number of applied sequences and/or increasing thedelay between them. For example, as shown below, a 1 min application hasan effect even after 15 minutes. Thus, it is expected that a shortapplication, for example, 20-60 seconds can be used to maintain morenormalized phosphorylation levels for many minutes, for example, 15, 20,40, 60 minutes or more. Optionally, a small number of spurts can thus beused to maintain relatively “normalized” levels for many hours, such as1, 2, 4, 6, 10, 12, 24 or more per hour (e.g., one spurt for each beator small number of beats such as 2, 5, 10, 20 or intermediate numbers).It should be noted that reduced frequency of application reduces totalpower needs.

In an exemplary embodiment of the invention, the delays between spurtsare not electric field free. For example, a continuous low level filedmay be applied, for example for causing hyper polarization of thetissue, reducing contractility and/or modifying immediate conductionvelocity. Similarly, a pulse sequence may maintain a baseline signal(constant or varying) even between pulses of the sequence. Optionally,the base line signal is selected to be non-excitatory, for example, byvirtue of its frequency (e.g., too high or too low) and/or power level.As noted herein, such a baseline signal may generate enough charge topositively affect phosphorylation.

In an exemplary embodiment of the invention, different stages intreatment are identified: immediate, mRNA, protein and physicalremodeling. Each state has its own time constants for initiation and fordecline. In an exemplary embodiment of the invention, a therapy isapplied long enough to have a noticeable effect on the stage whereeffect is required. Times of no therapy application may be selected tomatch the stage. For example, at remodeling stage, delays (noapplication times) of days or weeks may be acceptable. Optionally, evenat such a remodeling stage, applications to continue a lower level stage(e.g., immediate) may be applied, at least part of the time. In anexemplary embodiment of the invention, a complete therapy plan mayinclude desired effects on the different stages and an overlap ofsequences and sequence application times is generated to address thestages required at the point in the process required. Optionally, amodel is created linking the different stages and therapy application.This model is used to decide when therapy may be applied and when itmust be applied. Further, additional therapies may be evaluated forapplication depending on how they impact the overall strategy. In somecases, it is expected that computer modeling and/or such mechanisms willbe required to find a useful solution for a give case.

In some embodiments, power is lower to reduce battery requirement, toprevent noticeable effects by a patient and/or prevent immediate tissueeffects. Optionally, power is reduced for safety or comfort reasons, forexample, by selecting a power level that does not affect other tissue orparts of tissue than the tissue for which therapy is desired.

In an exemplary embodiment of the invention, the comfort factorconsidered is pain caused by inadvertent stimulation of nerves. In anexemplary embodiment of the invention, pain is reduced and/or avoided,by selecting a subset of a plurality of leads, electrodes and/or powersettings which minimizes pain and/or discomfort, while still providinguseful therapy (e.g., increase phosphorylation). Alternatively oradditionally, what is minimized is stimulation of the autonomous nervoussystem. Optionally, stimulation parameters are changed, as the patienthabituates. In an exemplary embodiment of the invention, the effect of asignal is estimated by calculating the field which will reach targettissue and base don tables which link the field strength on effects.Optionally, the tables are generated using tissue homogenate and/orextracted cardiomyocytes. Optionally, the testing is on the patient'sown tissue.

In an exemplary embodiment of the invention, pulse duration is in therange of 5-150 msec, for example, 10-40 msec. Alternatively pulseduration may be of one or combined groups in ranges such as 1 nsec-0.5usec, 0.1-10 usec, 1-100 usec, 10-500 usec, 100-1000 usec, 500 usec-10msec, 1-100 msec, 10-1000 msec, 100-10000 msec.

Additional exemplary pulse properties are now described.

Applied signals optionally are composed of such pulses, and theircombinations. Signals may also be applied to excitatory tissue withinthe refractory period of such tissue, or outside such refractory period,or during a relative refractory period extending after the end of suchperiod. Signals may be composed of pulses, square, saw-tooth, smooth orcontinuous waveforms, whether applied in stand alone pulses, in series,continuously, or superimposed.

Signals may be applied on every tissue activation cycle, e.g. everyheart beat, or other physiological cycle (breathing, sympathetic andparasympathetic systems cycle, other muscle contraction, etc).Alternatively, the application may be intermittently in only some of thecycles. Alternatively, signals may be applied at random timing or at apre-determined timing.

Treatment period may last, for example seconds, minutes, hours, days,weeks, months, or years.

Treatment may be alternating such as to be applied for some time period,and some rest period intermittently. For example, such treatmentschedule may be activated for several hours (e.g. about 1-5 hours) everyseveral hours (e.g. every 3-24 hours), or activated 12 hours out ofevery 24 hours (12 continuous hours every day or 12 intermittent hours:1 hour on followed by 1 hour off).

Treatment schedule may be configured for alternating among days orweeks, such as 3 days of treatment followed by 2 days of rest,repeatedly, or 4 weeks of treatment followed by 1 week of rest.

Alternatively, signals may be applied to provide treatment in a mannerthat changes according to changes in physiological condition. Suchchange in physiological may be sensed by the device, or may becommunicated to the device.

Signals may be selected from a group of signals, each with propertiesselected for a desired effect, and the device may alternate among thesignals, superimpose two or more of the signals, automatically adjustone or more of the signals, and/or change the ratio among the deliverytime and magnitude of those signals.

Genes and Related Proteins

Following is a partial list of genes (and corresponding proteins) whoseexpression is correlated with some types of heart failure (termed “heartfailure” in short below). In an exemplary embodiment of the invention,treatment is configured so that a particular gene/protein will beaffected in a desirable manner. It is noted that in accordance with someembodiments of the invention, different heart failure states areidentified based on the protein expression, mRNA expression and/orprotein activity profiles; or based on changes in such profiles inresponse to treatment, for example, immediate response or longer termresponse (e.g., hours, weeks or months). The treatment may targetparticular proteins, pathways or groups of proteins, for example, SRproteins. Optionally, the treatment aims to undo the negative effectsdescribed below, for example, by modifying the protein level and/oractivity. Optionally, analysis and/or treatment relates simultaneouslyto several genes, for example a set of two, a set of three, a set offive or sets of other numbers of genes, for example genes selected formthe list below. Optionally, the set includes genes from at least two orfrom at least three different gene type classifications. A profile usedfor assessment can include, for example, 1, 2, 3, 4, 5 or more markersfrom each of the types of mRNA, protein and protein activity.

Various Genes

a) ANP=atrial natriuretic peptide or A-type natriuretic peptide, isincreased in heart failure. The increase in ANP is related to increasedatrial enlargement and stretch which is bad. Increased ANP correlateswith increased mortality and morbidity in heart failure.

b) BNP=Brain natriuretic peptide or B-type natriuretic peptide isincreased in heart failure. BNP is elaborated from ventricular tissue.The increase in BNP is due largely to increased LV size and stretchwhich is bad in heart failure. Increased BNP in heart failure correlateswith increased mortality and/or morbidity. BNP is also a member of theso-called “fetal gene program” which also negatively impacts heartfailure.

c) GAPDH. This is a gene whose expression does not change in heartfailure and is used as “a housekeeping gene” to ensure good quality RNAextraction. If the expression of this gene changes during RT-PCR thismay indicate poor RNA quality and the results of all other geneexpression measurements may become questionable.

SR Genes

d) RYR2=ryanodine receptors also referred to as sarcoplasmic reticulumcalcium release channels. These channels control the release of calciumfrom the sarcoplasmic reticulum. This is the calcium signal that isneeded to activate the contractile apparatus (actin myosin crossbridging). These channels are hyperphosphorylated in heart failure andturn very active and, therefore, are “leaky,” leading to possiblecalcium overload which is bad for the heart muscle cell. Reducing ornormalizing phosphorylation may be desirable for these proteins.

e) NCX=sodium calcium exchanger. Under normal conditions, the NCX takescalcium out of the cell in return for Na. This maintains calciumhomeostasis and prevents calcium overload which is bad for muscle cellfunction and survival. In heart failure the NCX is increased and ishyperphosphorylated and may begin to work in what is called “reversemode”, to compensate for reduced SERCA-2A activity, and may causecalcium overload (=diastolic dysfunction). Too much activity in forwardmode depletes SR calcium (=systolic dysfunction).

f) PLB=Phospholamban. This is an essential sarcoplasmic reticulumprotein. Under normal conditions PLB is phosphorylated (PLB-P). Whenthat happens it activates SERCA-2a (calcium ATPase) which then pumpscalcium from the cytosol into the SR and thus prevents calcium overload.PLB is decreased in heart failure and is dephosphorylated. Because ofthat, SERCA-2a activity is reduced and it is less able to pump calciumback into the sarcoplasmic reticulum. This leads to calcium overload.When the SR has reduced calcium, there is less calcium release throughthe calcium release channels and contractility decreases.

g) SERCA-2a=calcium ATPase. This sarcoplasmic reticulum (SR) pump, undernormal conditions, pumps calcium from the cytosol into the SR. In heartfailure SERCA-2a decreases dramatically and its activity also decreasesleading to calcium overload and poor intracellular calcium cycling. Thisdecreases contraction strength.

h) Calsequestrin (CSQ). Clasequestrin is an SR protein involved incalcium sequestration and does not change in heart failure. Because itdoes not change in heart failure, it is frequently used as ahousekeeping gene. It may also be used to normalize when samples areinconsistent in the loading process.

Matrix Metalloproteinases (MMPs)

i) MMP 1. This gene is involved in the degradation of connective tissueat all levels and, for this reason, its elevation in heart failure isnot desirable and counteracted in some embodiments of the invention.

j) MMP2 and MMP9. These are referred to as to as “gelatinases”.Inhibiting gelatinases in the setting of heart failure appears to behelpful particularly with respect to reducing “interstitial fibrosis” orthe accumulation of connective tissue or collagen in the cardiacinterstitium. Reducing interstitial fibrosis leads to improved LVdiastolic compliance and, hence, improved diastolic filling andfunction.

Stretch Response Genes

Stretch response genes are up-regulated in the presence of progressiveLV dilation and myocytes stretch as occurs in heart failure. Theimportance of these genes is they trigger maladaptive cardiomyocyteshypertrophy which then leads to abnormal calcium cycling.

k) p38=p38 alpha-beta mitogen activated protein kinese. This is astretch response gene. Its expression increases in heart failure andthat can lead to many abnormalities including the development ofhypertrophy and activation of multiple transcription factors that leadto activation of the fetal gene program. An increase in P38 correlateswith maladaptive hypertrophy and ventricular enlargement. This indicatesa bad prognosis for heart failure.

l) p21 ras=This is also a stretch response gene. Its expressionincreases in heart failure due to ventricular enlargement and stretch.When stretch and wall stress increases in heart failure, thesemechanical factors increase a family of cell surface proteins known asintegrins. Integrins, when activated, lead to increase in p21 ras andp38 and both lead to maladaptive hypertrophy.

m) Integrin-a5. This is a cell surface receptor gene whose protein actsas a mechanical transducer. It is activated in response to mechanicalstretch or stress mediated by LV dilation. When activated, it promotesregulation of stretch response protein. Down regulation of this gene inheart failure is a desirable feature of some embodiments of theinvention.

Fetal Program Genes

n) Alpha-MHC=alpha myosin heavy chain is reduced in heart failure.Because the alpha isoform of MHC is the isoform responsible forincreased velocity of shortening of cardiac muscle cells, a reduction inalpha MHC impacts negatively on function/contraction of the failingventricle. Alpha MHC restoration is associated (and optionally providedby some embodiments of the invention) with LV contraction improvements.

o) Beta1-Adrenergic Receptor. This gene is down-regulated in heartfailure. Drugs such as metoprolol which are selective beta-1 receptorblockers which up-regulate the beta-1 receptor improve mortality andmorbidity in heart failure and appear to also improve, albeit in alimited way, exercise tolerance. Up-regulation of this gene is viewed asa positive development in some embodiments of the invention whichenhances the sensitivity of the contractile element of catecholamines.

SERCA-2a, mentioned above, is also a member of the so-called fetalprogram gene.

Calcium Binding Proteins

Enhanced expression of calcium binding proteins such as S100A1 andsorcin improve contractility by increasing calcium uptake into thesarcoplasmic reticulum. Expression of these proteins can also modify thebehaviors of ryanodine calcium release channels. Alternativelydown-regulation (or avoiding upregulation) of these calcium bindingproteins can diminish contractility. This may be applicable tohypercontractile states of the heart associated with certain diseasessuch as hypertrophic obstructive cardiomyopathy. Contractility may alsobe reduced using hyperpolarizing fields.

Experimental Results—Long Term (“Chronic”) and Short Term (SeveralHours)

Before describing experiments in which an immediate effect wasdiscovered on phosphorylation, experiments on relatively long termeffects will be described, including effects that occur (at least to asignificant degree) after a few hours of continuous CCM application andafter 3 months. These experiments generally show that LV function indogs with HF improves without an associated increase in MVO₂.

In this experimental preparation, chronic HF is produced by multiplesequential intracoronary embolizations with polystyrene Latexmicrospheres (70-102 μm in diameter) which result in loss of viablemyocardium, LV enlargement and a decrease in LV ejection fraction. 14healthy mongrel dogs weighing between 20 and 30 kg underwent coronarymicroembolizations to produce HF. Embolizations were performed one weekapart and were discontinued when LV ejection fraction, determinedangiographically, was <30%. Microembolizations were performed duringcardiac catheterization under general anesthesia and sterile conditions.Animals were induced with intravenous oxymorphone hydrochloride (0.22mg/kg) and diazepam (0.17 mg/kg) and a plane of anesthesia wasmaintained with 1-2% isoflurane. Some of the results shown herein usefewer than all dogs.

Two weeks after the target LV ejection fraction was reached, dogs wereanesthetized as described above, intubated and ventilated with room air.The right external jugular vein was surgically exposed and used toposition the CCM leads. Two standard active fixation leads were advancedinto the right ventricle and positioned on the anterior and posteriorseptal grooves and were used to sense ventricular activity and deliverCCM electrical signals. A third active fixation lead was positioned inthe right atrium for p-wave sensing. The leads were connected to a CCMsignal generator (OPTIMIZER™ II, Impulse Dynamics NV, Curacao Nev.). Thegenerator was implanted in a subcutaneous pocket created on the rightside of the neck. All 14 animals were implanted and all were allowed torecover. Studies were performed 2 weeks after OPTIMIZER™ Systemimplantation. This period of time was allowed to ensure that the leadsstabilized in place.

Two weeks after OPTIMIZER™ System implantation, dogs were anesthetizedand underwent a pre-treatment left and right heart catheterization toassess hemodynamics and measure MVO₂. Dogs were then randomized to anactive treatment group (n=7) or to a sham-operated control group (n=7).In the active treatment group, the OPTIMIZER™ system was activated todeliver CCM therapy. CCM therapy was administered for 5 hours/day basedon a duty cycle of one hour ON (CCM signal±7.73 volts) and 3 hours and48 minutes OFF for 3 months. Sham-operated control dogs did not receiveany therapy whatsoever and were also followed for 3 months. At the endof 3 months of therapy or follow-up, all hemodynamic measures wererepeated including MVO₂. After completion of all hemodynamicmeasurements and while under general anesthesia, the dogs' chest wasopened and the heart rapidly harvested and tissue from theinter-ventricular septum and LV free wall was obtained and prepared forhistological and biochemical evaluation. Tissue from 6 normal dogs wasobtained and prepared in the same manner and used for comparisons. Alltissue was stored at −70° C. until needed.

To address certain aspects of the mechanisms of action of CCM therapy, aseries of 6 additional dogs underwent intracoronary microembolizationsto produce HF as described earlier. In these dogs, under generalanesthesia, a mid-sternotomy was performed, the pericardium was openedand epicardial CCM leads were placed on the anterior wall between the2nd and 3rd diagonal branches. Hemodynamic measurements includingmeasurements of MVO₂ were made before and 2 hours after continuous CCMsignal delivery at 7.73 volts. At the end of 2 hours of therapy,myocardial samples were obtained from the LV anterior wall in the regionof the CCM leads and from the LV posterior wall remote from the CCMleads. Left ventricular tissue from 6 normal dogs and 6 HF dogs thatwere untreated was obtained and prepared in the same manner and used forcomparisons. All tissue was rapidly frozen in liquid nitrogen and storedat −70° C. until needed.

Aortic and LV pressures were measured with catheter-tip micromanometers(Millar Instruments, Houston, Tex.) during cardiac catheterization. LVend-diastolic pressure was measured from the LV waveform. Single-planeleft ventriculograms were obtained during each catheterization aftercompletion of the hemodynamic measurements with the dog placed on itsright side. Ventriculograms (approximately 60 right anterior obliqueprojection) were recorded on 35 mm cine film at 30 frames per secondduring the injection of 20 ml of contrast material (Reno-M-60, Squibb,Princeton, N.J.). Correction for image magnification was made with aradiopaque calibrated grid placed at the level of the LV. LVend-diastolic volume and end-systolic volume were calculated fromventricular silhouettes using the area-length method, such as describedin Dodge H T, Sandler H, Baxley W A, Hawley R R. Usefulness andlimitations of radiographic methods for determining left ventricularvolume. Am J. Cardiol. 1966; 18:10-24, the disclosure of which isincorporated herein by reference. Stroke volume was calculated as thedifference between LV end-diastolic volume and end-systolic volume.Total coronary blood flow (CBF), and MVO₂, were measured and calculatedas described, for example, in Chandler M P, Stanley W C, Morita H,Suzuki G, Roth B A, Blackburn B, Wolff A, Sabbah H N. Acute treatmentwith ranolazine improves mechanical efficiency in dogs with chronicheart failure. Circ Res. 2002; 91:278-280, the disclosure of which isincorporated herein by reference. LV mechanical efficiency wascalculated as the ratio of LV power to MVO₂ following the same paper.

Calsequestrin (CSQ), atrial natriuretic peptide (ANP), brain natriureticpeptide (BNP), ryanodine receptor (RyR), total phospholamban (PLB),phosphorylated PLB (P-PLB), sarcoplasmic reticulum (SR) calcium ATPase(SERCA-2a), and β₁-adrenergic receptor (β₁-AR) were measured by WesternBlots. Briefly, LV homogenate was prepared from ˜100 mg LV powder asdescribed, for example, in Gupta R C, Mishra S., Mishima T, Goldstein S,Sabbah H N. Reduced sarcoplasmic Reticulum Ca²⁺-uptake and PhospholambanExpression in Ventricular Myocardium of Dogs with Heart Failure. J MollCell Cardiol. 1999; 31:1381-1389, the disclosure of which isincorporated herein by reference and protein determined using the Lowrymethod, for example as described in Lowry O H, Rosebrough N J, Farr A L,Randall R J. Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature. 1951; 27:680-685, the disclosure ofwhich is incorporated herein by reference. CSQ, a calcium-bindingprotein located in the SR and is unchanged in HF was used to normalizeprotein loading on the gel. Approximately 20-100 μg protein of eachsample was separated on 4-20% SDS-polyacrylamide gel and the separatedproteins were electrophoretically transferred to a nitrocellulosemembrane. The accuracy of the electrotransfer was confirmed by stainingthe membrane with 0.1% amido black. For identification of the desiredprotein, the nitrocellulose blot was incubated with the appropriatelydiluted primary monoclonal or polyclonal antibody specific to eachprotein based on the supplier's instructions. Antibody-binding proteinswere visualized by autoradiography after treating the blots withhorseradish peroxidase-conjugated secondary antibody (anti-rabbit) andECL color developing reagents. ANP, BNP, β₁-AR, SERCA-2a, PLB, orRyR-specific antibody recognized 20, 14, 65, 110, 5.5, and 250 kDaprotein bands respectively. Total P-PLB was quantified inSDS-phosphoprotein-enriched fraction (PPE) prepared from LV homogenateusing PLB specific monoclonal antibody. P-PLB at serine-16 orthreonine-17 was quantified in SDS-LV homogenate using primaryantibodies specific to the P-PLB at serine-16 or at threonine-17. PPEwas prepared from LV tissue using a BD Bioscience phosphoproteinenrichment kit, for example as described in Gupta R C, Mishra S, Yang XP, Sabbah H N. Reduced inhibitor 1 and 2 activity is associated withincreased protein phosphatase type 1 activity in left ventricularmyocardium of one-kidney, one-clip hypertensive rats. Mol Cell Biochem.2005; 269:49-57, the disclosure of which is incorporated herein byreference. Band intensity was quantified using a Bio-Rad GS-670 imagingdensitometer and expressed as densitometric units×mm². In all instances,the antibody was present in excess over the antigen and the density ofeach protein band was in the linear scale.

mRNA expression of glyseraldehyde-3-phosphate dehydrogenase (GAPDH),α-myosin heavy chain (MHC), β₁-AR, ANP, BNP, SERCA-2a, total PLB, RyRand CSQ was measured. Total RNA with an absorbance ratio (260 nm/280 nm)above 1.7 was isolated from frozen LV tissue as described in Mol CellBiochem. 2005; 269:49-57. Approximately 4-10 μg RNA wasreverse-transcribed into cDNA in an assay volume of 80 microliter. Foreach polymerase chain reaction (PCR), 2-5 μl first-strand cDNA was addedto 50 μl of a reaction mixture containing 20 pmol of each forward andreverse primer of each gene, 200 μl of each dNTP, 10 mM Tris-HCl (pH8.8), 50 mM KCl, 0.1% Triton-X100 and 3.0 mM MgCl₂ and 1 unit platinumTaq DNA polymerase (Invitrogen, Carlsbad, Calif.) and PCR allowed toproceed for 20 to 40 cycles. For each gene, PCR cycle was determined toensure that the gene product is forming in linear range. PCR productswere analyzed by subjecting 20 μl of each reaction mixture toelectrophoresis on 1%-1.5% ethidium-bromide-agarose gels. Band size ofthe products was compared with standard DNA size markers and confirmedby sequencing of the forward (F) and reverse (R) primers for each geneand gene product. mRNA expression of α-MHC was measured by amplificationof cDNA by reverse transcriptase-PCR followed by digestion with Pst1restriction enzyme as described, for example, in Feldman A M, Ray P E,Silan C M, Mercer J A, Minobe W, Bristow M R. Selective gene expressionin failing human heart: quantification of steady-state levels ofmessenger RNA in endomyocardial biopsies using the polymerase chainreaction. Circulation. 1991; 83: 1866-1872, the disclosure of which isincorporated herein by reference. Fluorescent band intensity wasquantified using a Bio-Rad GS-670 imaging densitometer and expressed asoptical density×mm².

From each heart, 3 transverse slices (approximately 3 mm thick) one eachfrom basal, middle and apical thirds of the LV, were obtained. Forcomparison, tissue samples obtained from 7 normal dogs were prepared inan identical manner. From each slice, transmural tissue blocks wereobtained and embedded in paraffin blocks. From each block, 6 μm thicksections were prepared and stained with Gomori trichrome to identifyfibrous tissue. The volume fraction of replacement fibrosis, namely, theproportion of scar tissue to viable tissue in all three transverse LVslices, was calculated as the percent total surface area occupied byfibrous tissue using computer-based video densitometry (MOCHA, JandelScientific, Corte Madera, Calif.). Transmural tissue blocks wereobtained from the free wall segment of the slice, mounted on cork usingTissue-Tek embedding medium (Sakura, Torrance, Calif.), and rapidlyfrozen in isopentane pre-cooled in liquid nitrogen and stored at −70° C.until used. Cryostat sections were prepared and stained withfluorescein-labeled peanut agglutinin (Vector Laboratories Inc.,Burlingame, Calif.) after pretreatment with 3.3 U/ml neuraminidase typeV (Sigma Chemical Co., St. Louis, Mo.) to delineate the myocyte borderand the interstitial space, including capillaries, for example asdescribed in Liu Y H, Yang X P, Sharov V G, Nass O, Sabbah H N, PetersonE, Carretero O A. Effects of angiotensin-converting enzyme inhibitorsand angiotensin II type 1 receptor antagonists in rats with heartfailure. Role of kinins and angiotensin II type 2 receptors. J ClinInvest. 1997; 99:1926-1935, the disclosure of which is incorporatedherein by reference. Sections were double stained with rhodamine-labeledGriffonia simplicifolia lectin I (GSL I) to identify capillaries. Tenradially oriented microscopic fields (magnification×100, objective×40,and ocular 2.5), were selected at random from each section andphotographed using 35 mm color film. Fields containing scar tissue(infarcts) were excluded. Average cross-sectional area of each myocytewas measured using computer-based planimetry. The volume fraction ofinterstitial collagen (reactive interstitial fibrosis) was calculated asthe percent total surface area occupied by interstitial space, minus thepercent total area occupied by capillaries (following J Clin Invest.1997; 99:1926-1935). Capillary density was calculated as the number ofcapillaries per square millimeter and as the index capillary per fiberratio. The oxygen diffusion distance was measured as half the distancebetween two adjoining capillaries.

To ensure that all hemodynamic measures were similar at baseline,comparisons were made between the two study groups before anyembolization and at the time of randomization before initiating activetherapy. To assess treatment effect, the change in each measure frompre-treatment to post-treatment was calculated for each of the studygroups and then compared between groups. For these comparisons at-statistic for two means was used with a probability value≦0.05considered significant. Within group comparisons between pre-treatmentand post-treatment hemodynamic measures were made using a Student'spaired t-test with p≦0.05 considered significant. Histomorphometric,biochemical and molecular differences between normal dogs, sham-operateduntreated HF dogs and CCM-treated HF dogs were examined using one-wayanalysis of variance (ANOVA) with a set at 0.05. If significance wasachieved, pairwise comparisons were performed among groups using theStudent-Newman-Kuels test with a probability value of ≦0.05 consideredsignificant. All hemodynamic, ventriculographic and histomorphometricassessments were made by investigators blinded to treatment group.

The results of these experiments are presented in summary below and ingreater detail after.

At baseline, all dogs entered into the study had hemodynamic measuresthat were within normal limits for conditioned mongrel dogs. Thehemodynamic and ventriculographic results obtained before initiatingtherapy or follow-up (pre-treatment) and 3 months after initiatingtherapy or follow-up (post-treatment) are shown in Table 1 for dogsrandomized into the CCM-active treatment group and Table 2 for dogsrandomized into the sham-operated untreated HF group. In sham-operateddogs, comparison of pre-treatment to post-treatment showed nodifferences in heart rate, systolic aortic pressure, LV end-diastolicpressure or stroke volume (Table 1). In this group, LV end-diastolic andend-systolic volume increased significantly while LV ejection fractiondecreased significantly. This was associated with an increase in totalCBF and an increase in MVO₂. LV mechanical efficiency tended to decreasebut the reduction was not statistically significant (Table 1).

TABLE 1 Chronic Hemodynamic and Angiographic Findings in Sham-OperatedDogs with Heart Failure Before (Pre-Treatment) and After 3 Months ofFollow-up (Post-Treatment) (n = 7) Pre-Treatment Post-Treatment P-ValueHeart Rate (beats/min) 89 ± 4 101 ± 7 NS Systolic Aortic Pressure (mmHg)97 ± 6 104 ± 6 NS LV EDP (mmHg) 13 ± 1  14 ± 1 NS LV EDV (ml) 67 ± 2  77± 2 0.0001 LV ESV (ml) 49 ± 2  60 ± 2 0.0001 LV EF (%) 27 ± 1  23 ± 10.001  Stroke Volume (ml) 18 ± 1  17 ± 1 NS Total LV CBF (ml/min), n = 642 ± 4  59 ± 6 0.015  MVO₂ (μmols/min), n = 6 193 ± 25  286 ± 36 0.026 LV Efficiency (%), n = 6 28 ± 4  21 ± 3 NS LV = left ventricular; EDP =end-diastolic pressure; EDV = end-diastolic volume; ESV = end-systolicvolume; EF = ejection fraction; CBF = coronary blood flow; MVO₂ =myocardial oxygen consumption. P-Value = Probability value based oncomparison between Pre- and Post-Treatment. NS = Not significant.

In CCM-treated dogs, comparison of pre-treatment to post-treatment alsoshowed no differences in heart rate and systolic aortic pressure. LVend-diastolic pressure decreased as did LV end-diastolic volume andend-systolic volume while stroke volume and LV ejection fractionincreased (Table 2). This functional improvement was associated with adecrease in total CBF and a decrease in MVO₂ along with a significantincrease in LV mechanical efficiency (Table 2).

TABLE 2 Chronic Hemodynamic and Angiographic Findings in CCM-TreatedDogs with Heart Failure Before (Pre-Treatment) and 3 Months AfterInitiating Treatment (n = 7) Pre-Treatment Post-Treatment P-Value HeartRate (beats/min) 78 ± 3 87 ± 3 NS Systolic Aortic 101 ± 4  102 ± 5  NSPressure (mmHg) LV EDP (mmHg) 14 ± 1  8 ± 1 0.01 LV EDV (ml) 70 ± 6 66 ±5 0.08 LV ESV (ml) 52 ± 6 45 ± 4 0.008 LV EF (%) 27 ± 1 33 ± 1 0.0001Stroke Volume (ml) 18 ± 1 21 ± 1 0.0001 Total LV CBF (ml/min) 73 ± 7 43± 3 0.005 MVO₂ (μmols/min) 275 ± 39 168 ± 19 0.01 LV Efficiency (%) 19 ±4 36 ± 5 0.0001

The change (Δ) in hemodynamic and ventriculographic measures frompre-treatment to post-treatment was compared between the two studygroups (Table 3). Heart rate and systolic aortic pressure wereunchanged. Compared to sham-operated controls, CCM-treated dogs had asignificantly lower LV end-diastolic pressure, end-diastolic volume andend-systolic volume along with significantly higher LV ejection fractionand stroke volume (Table 3). This improvement in LV function inCCM-treated dogs was accompanied by a significant reduction of CBF andMVO₂ and a significant increase in LV mechanical efficiency (Table 3).

TABLE 3 TREATMENT EFFECT Comparison of the Change (Δ) from Pre-treatmentto Post-Treatment between Sham-Operated Untreated Heart Failure Dogs andCCM-Treated Heart Failure Dogs Sham-Operated CCM-Treated Untreated HFDogs HF Dogs P-Value Heart Rate (beats/min) 12 ± 8  8 ± 4 NS SystolicAortic 6 ± 6 1 ± 7 NS Pressure (mmHg) LV EDP (mmHg) 1 ± 2 −6 ± 2  0.029 LV EDV (ml) 10 ± 1  −4 ± 2  0.0001 LV ESV (ml) 11 ± 1  −7 ± 2  0.0001 LVEF (%) −4 ± 1  6 ± 1 0.0001 Stroke Volume (ml)  −1 ± 0.5   3 ± 0.40.0001 Total LV CBF (ml/min), n = 6 18 ± 5  −30 ± 7  0.0001 MVO₂(μmols/min), n = 6 93 ± 30 −96 ± 27  0.001  LV Efficiency (%), n = 6 −7± 4  16 ± 2  0.0001 P-Value = Probability value based on comparisonbetween sham-operated and CCM-treated dogs. Other abbreviations are thesame as in table 1.

Histomorphometric results are shown in Table 4. Volume fraction ofreplacement fibrosis, volume fraction of interstitial fibrosis andcardiomyocyte cross-sectional area were significantly higher insham-operated dogs compared with normal dogs. Volume fraction ofreplacement fibrosis was reduced by 23%, volume fraction of interstitialfibrosis was reduced by 16% and average cardiomyocyte cross-sectionalarea was reduced by 19% compared to sham-operated HF dogs. Capillarydensity decreased in sham-operated HF dogs while oxygen diffusiondistance increased when compared to normal dogs (Table 4). CCM therapyrestored capillary density and oxygen diffusion distance to near normallevels (Table 4).

TABLE 4 Chronic Histomorhometric Findings in Left Ventricular Myocardiumof Normal Dogs, Sham-Operated Untreated Heart Failure Dogs andCCM-Treated Heart Failure Dogs Normal Sham-Operated CCM-Treated DogsUntreated HF Dogs HF Dogs VFRF (%) 0.0 14.3 ± 1.5* 11.0 ± 0.8*† VFIF (%)3.7 ± 0.1 11.2 ± 0.3*  9.4 ± 0.7*† MCSA (μm²) 409 ± 10  719 ± 36* 581 ±28*† CD (# capillaries/mm²) 2607 ± 80   1882 ± 67*  2192 ± 117*† CD (#capillaries/fiber) 1.00 ± 0.0   0.92 ± 0.02*  1.03 ± 0.02*† ODD (μm) 8.9± 0.2 11.7 ± 0.3* 10.2 ± 0.2*† VFRF = Volume fraction of replacementfibrosis; VFIF = Volume Fraction of Interstitial Fibrosis; MCSA =Cardiomyocyte cross-sectional area; CD = Capillary density; ODD = Oxygendiffusion distance. *= p < 0.05 vs. normal dogs; †= p < 0.05 vs.Sham-operated dogs.

In a set of short term experiments, 6 dogs that had micro-embolizationinduced HF were used. The CCM signal was a 7.73 volt, epicardial LVanterior wall signal. The definitions for the various variables followCirculation Research 91:278, 2002, the disclosure of which isincorporated herein by reference. The signal is applied to theepicardial surface of the heart.

Where not otherwise specified, CCM is a pulse at 80 applications perminute (synchronized to a heart beat, if any), at 7.73 Volts, withbetween 4 and 6 phases, each phase being 5.56 ms long and beingcontinuous and of opposite polarity of a previous phase. The number ofphases was not changed within an experiment. The signal was generallyapplied to a septum, from a right chamber, with a distance of 1-2 cmbetween an electrode pair used to apply the signal.

The marking “NL” indicates normal tissue levels.

FIGS. 2A-2C show the improvement of LV function without MVO₂ increase,in CCM treated dogs as a function of time and compared to baselinevalues.

FIGS. 2D-2G show changes in mRNA expression of alpha-MHC, ANP, BNP andSERCA-2a in normal, HF and HF dogs with CCM treatment respectively,after several hours of treatment.

As will be shown below, effects on protein phosphorylation (at least)can be shown after a short time.

FIG. 2H shows phosphorylated phospholamban normalized to totalphospholamban following therapy of several hours (indicated herein as“short-term”). FIG. 2I shows corresponding blots using a WesternBlotting method. It should be noted that both phospholamban levels andphosphorylation levels thereof improve with therapy.

FIG. 2J shows reduction in mRNA expression of NCX following CCMtreatment. FIG. 2K shows a general (or slight reduction) normalizationof NCX protein values while still maintaining increased relativephosphorylation. This may allow some compensation for disturbed cardiacfunction.

FIG. 2L shows decreased mRNA expression of GATA-4, to even below normallevels. FIG. 2M shows that protein expression of GATA-4, is however,still increased relative to normal. This may be useful for control ofNCX and/or other proteins. This result also indicates that merelycontrolling mRNA may not sufficiently determine the cellular behavior,as protein levels and/or phosphorylation levels may compensate or overcompensate. In general, however, the levels are normalized as comparedto HF.

FIGS. 2N and 2O show the effect of chronic (e.g., 3 month) treatmentwith CCM on mRNA expression profiles. Normalization of these importantproteins can be seen.

It should be noted that the electric field can operate differently ondifferent proteins, for example, directly affecting some proteins andthese proteins indirectly affecting the behavior and/or levels of otherproteins. It should also be noted that there are multiple pathways inthe cells and the electrical treatment may affect multiple (e.g., 1, 2,3, 4 or more) pathways in parallel. The resulting effects on proteinsmay be increasing or decreasing their expression and/or activity levels.Different such effects may be desirable for different proteins and/ordifferent disease conditions. Different proteins may be predisposed(e.g., based on their structure, surrounding materials and/or existingpathways) to differently increase and/or decrease. A particularexperiment with Phospholamban is described below.

The following tables summarize the results on mRNA expression for normaldogs (NL), dogs with HF and dogs with HF and chronic CCM treatment.Protein levels and phosphorylation levels are described later on. Asummarizing table is also provided later on in the application.

Dog Numbers HF-Control HF + CCM 02-097 02-106 02-098 02-107 02-10302-108 02-130 02-012 03-045 03-023 04-004 03-050 04-018 04-005

mRNA Expression for GAPDH NL HF-Control HF + CCM 223 195 246 227 195 223238 223 232 215 241 250 217 227 229 192 237 237 240 232 Mean 219 223 236STD 15 20 10 SEM 6 8 4 ANOVA = 0.16

mRNA Expression for TNF-alpha NL HF-Control HF + CCM 48 235 85 53 223117 36 182 107 28 194 98 39 232 144 31 234 81 240 92 Mean 39 220 103 STD10 23 22 SEM 4 9 8 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control<0.05

mRNA Expression for Activin-A NL HF-Control HF + CCM 101 302 174 104 295142 109 282 150 136 269 148 153 263 100 199 232 92 245 88 Mean 134 270128 STD 38 26 34 SEM 15 10 13 ANOVA = 0.0001 p vs. NL <0.05 NS p vs.HF-Control <0.05

mRNA Expression for Tubulin-alpha NL HF-Control HF + CCM 140 164 82 120160 124 125 162 144 117 185 146 124 165 160 125 176 141 163 168 Mean 125168 138 STD 8 9 28 SEM 3 3 11 ANOVA = 0.002 p vs. NL <0.05 NS p vs.HF-Control <0.05

mRNA Expression for ANP NL HF-Control HF + CCM 21 35 26 19 29 27 17 2729 12 28 27 18 38 28 23 35 26 43 24 Mean 18 34 27 STD 4 6 2 SEM 2 2 1ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control <0.05

mRNA Expression for TIMP-1 NL HF-Control HF + CCM 160 164 131 185 188146 184 243 136 203 235 191 248 185 151 270 130 170 173 174 Mean 208 188157 STD 42 40 22 SEM 17 15 8 ANOVA = 0.052

mRNA Expression for IL-6 NL HF-Control HF + CCM 277 556 430 302 533 409349 524 433 337 547 409 350 558 421 348 552 381 567 365 Mean 327 548 407STD 31 15 25 SEM 12 6 10 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs.HF-Control <0.05

mRNA Expression for Titin NL HF-Control HF + CCM 183 137 281 172 222 278241 182 242 285 197 224 313 124 257 294 135 205 196 231 Mean 248 170 245STD 60 38 28 SEM 24 14 11 ANOVA = 0.005 p vs. NL <0.05 NS p vs.HF-Control <0.05

mRNA Expression for Tubulin-beta NL HF-Control HF + CCM 88 123 75 77 12779 68 115 94 65 146 91 60 133 108 62 116 98 133 105 Mean 70 128 93 STD11 11 12 SEM 4 4 5 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control<0.05

mRNA Expression for BNP NL HF-Control HF + CCM 32 173 30 13 182 47 21166 59 31 173 56 22 194 35 17 186 25 163 58 Mean 23 177 44 STD 8 11 14SEM 3 4 5 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control <0.05

mRNA Expression for TIMP-2 NL HF-Control HF + CCM 233 252 222 200 247239 223 238 235 230 239 229 210 229 225 246 240 219 224 198 Mean 224 238224 STD 17 10 13 SEM 7 4 5 ANOVA = 0.12

mRNA Expression for MMP-1 NL HF-Control HF + CCM 24 40 29 20 38 26 18 3230 24 31 24 23 41 25 33 54 34 32 37 Mean 24 38 29 STD 5 8 5 SEM 2 3 2ANOVA = 0.003 p vs. NL <0.05 NS p vs. HF-Control <0.05

mRNA Expression for MMP-9 NL HF-Control HF + CCM 35 60 45 25 69 49 29 4349 26 71 29 31 44 42 27 41 64 39 42 Mean 29 52 46 STD 4 14 11 SEM 2 5 4ANOVA = 0.003 p vs. NL <0.05 <0.05 p vs. HF-Control NS

mRNA Expression for p38 MAPK NL HF-Control HF + CCM 48 41 40 52 60 44 2546 28 41 57 29 27 59 33 25 67 20 56 20 Mean 36 55 31 STD 12 9 9 SEM 5 33 ANOVA = 0.0001 p vs. NL <0.05 NS p vs. HF-Control <0.05

mRNA Expression for NCX NL HF-Control HF + CCM 25 85 31 30 115 41 36 6355 32 139 50 28 39 46 29 121 53 126 62 Mean 30 98 48 STD 4 37 10 SEM 214 4 ANOVA = 0.0001 p vs. NL <0.05 NS p vs. HF-Control <0.05

mRNA Expression for Beta1-receptor NL HF-Control HF + CCM 22 11 18 21 1017 20 6 17 19 8 18 24 12 19 25 11 22 13 17 Mean 22 10 18 STD 2 2 2 SEM 11 1 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control <0.05

mRNA Expression for MMP-2 NL HF-Control HF + CCM 25 56 42 22 42 29 24 4030 22 39 28 23 41 30 24 48 27 41 33 Mean 23 44 31 STD 1 6 5 SEM 0 2 2ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control <0.05

mRNA Expression for p21RAS NL HF-Control HF + CCM 85 284 297 88 295 270162 305 259 167 277 248 202 299 228 213 295 202 284 201 Mean 153 291 244STD 55 10 36 SEM 22 4 13 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs.HF-Control <0.05

mRNA Expression for Integrin-a5 NL HF-Control HF + CCM 7 17 7 10 14 9 414 10 11 15 7 8 25 6 7 20 10 12 16 Mean 8 17 9 STD 2 4 3 SEM 1 2 1 ANOVA= 0.0001 p vs. NL <0.05 NS p vs. HF-Control <0.05

mRNA Expression for GATA-4 NL HF-Control HF + CCM 83 153 84 142 247 103136 138 242 105 240 191 78 164 113 71 254 254 233 135 Mean 103 204 160STD 31 50 69 SEM 12 19 26 ANOVA = 0.012 p vs. NL <0.05 <0.05 p vs.HF-Control <0.05

mRNA Expression for SERCA-2a NL HF-Control HF + CCM 218 167 172 235 189200 242 159 194 238 178 192 250 178 195 232 179 198 171 193 Mean 236 174192 STD 11 10 9 SEM 4 4 3 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs.HF-Control <0.05

mRNA Expression for PLB NL HF-Control HF + CCM 221 170 217 229 149 224255 155 237 241 146 222 248 167 210 200 149 190 109 182 Mean 232 149 212STD 20 20 19 SEM 8 8 7 ANOVA = 0.0001 p vs. NL <0.05 NS p vs. HF-Control<0.05

mRNA Expression for CSQ NL HF-Control HF + CCM 261 274 192 258 271 225247 281 262 280 291 299 268 250 276 269 276 281 279 281 Mean 264 275 259STD 11 13 38 SEM 5 5 14 ANOVA = 0.47 p vs. NL NS NS p vs. HF-Control NS

mRNA Expression for RYR NL HF-Control HF + CCM 40 25 39 36 23 30 31 2915 42 18 33 34 21 34 42 22 58 17 43 Mean 38 22 36 STD 5 4 13 SEM 2 2 5ANOVA = 0.008 p vs. NL <0.05 NS p vs. HF-Control <0.05

mRNA Expression for α-MHC NL HF-Control HF + CCM 176 133 192 212 136 165219 115 158 218 140 181 221 179 176 224 192 194 144 192 Mean 212 148 180STD 18 27 14 SEM 7 10 5 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs.HF-Control <0.05Short Discussion of Some Results

Phospholamban is down regulated in heart failure and is nearlynormalized with CCM therapy. This may explain the improvement in LVfunction by CCM treatment. CCM appears to normalize the RYR messagewhich is consistent with proper therapy. The up-regulation of alpha-MHCwith CCM may be contributing to the sustained long-term improvement inLV ejection fraction. Decrease in MMP1 following CCM therapy is in andof itself desirable. Inhibiting gelatinases, as shown, is beneficial,possibly reducing interstitial fibrosis and leading to improved LVdiastolic compliance and, hence, improved diastolic filling andfunction. p21RAS and p38 mitogen activated protein kinese (MAPK) areemissions of stretch response genes which are down-regulated followingCCM therapy and correlate with reduced cardiomyocytes hypertrophy.Integrin-a5 is clearly normalized following long-term CCM therapy.Up-regulation of Beta1-Adrenergic Receptor is viewed as a positivedevelopment which enhances the sensitivity of the contractile element ofcatecholamines. Beta-blockers are known to enhance the sensitivity ofthe myocardium to exogenous as well as endogenous catecholamines whenused in heart failure patients over long periods of time in excess of 3months. In the normal heart, beta blockers reduce the sensitivity of theheart to catecholamines. As will be described below, the therapies asdescribed herein may be used together with beta-blockers, for both shortand long term synergistic effects.

Possibly, some therapies according to the present invention will focuson general improvement of health, while other therapies will focus onincreasing tissue responsiveness, for example, to certain drugs, andthus focus on improving fewer than all mRNA and/or protein and/orphosphorylation indicators.

Experimental Results—Immediate

The inventors of the present application have discovered that,surprisingly, phosphorylation effects for at least some proteins can begenerated in immediate time frames, such as less than 1 minute and evenless than 10 or 5 seconds in some cases. Further, an immediacy of effectis also characterized by a reduced number of intermediate stages,indicated by the fact that protein phosphorylation effects can beimposed even in tissue homogenate. Further, specificity of thephosphorylation effects to certain proteins that are relevant for HF isalso shown. Further, a lack of effect of an exemplary pacing signal isalso shown.

Tissue Homogenate

FIGS. 3A and 3B show the effect of a CMM signal applied to tissuehomogenate from LV failed heart tissue. As can be seen, even a 10 secondsignal was sufficient to generate noticeable and significant changes inphosphorylation. Changes in phosphorylation are shown in Serine-16Monometric PLB (Phospholamban) form, Threonin-17 mometric PLB form,Serine 16 Pentametric PLB form and Ryanodine channels.

The tissue homogenate was prepared in the following manner.Approximately 14 g frozen LV tissue from a dog with chronic HF in 42 ml50 mM Tris-HCl, pH 7.5 was homogenized three times for 20 seconds eachtime using a 10-mm generator (Omni International, Waterbury, Conn.) atsetting 10. The homogenate was then filtered through 4 layers of cheesecloth. The resulting homogenate was stored in ice and its proteinconcentration was determined by the Lowry method.

The CCM signals were delivered to the homogenate as follows. Thehomogenate was diluted 2 fold in homogenate buffer and subsequentlyaliquotted 3 ml each in assay tubes. Assay tubes were divided into 2sets (Set A and Set B), each subset consisting of 7 assay tubes. CCMsignals were delivered for 10″, 30″, 1′, 5′, 30′, and 60′ in one of thesets, while the other set served as time control. The reaction wasstopped by adding concentrated SDS. Protein assay on all the sampleswere performed by Lowry method.

Phosphorylation of PLB at serine-16 (Ser-16) and threonine-17 (Thr-17)was determined by Western blotting using specific antibody as describedin Mishra S, Sabbah H N, Jain J C, Gupta R C: “ReducedCa2+-calmodulin-dependent protein kinase activity and expression in LVmyocardium of dogs with heart failure”, Am J Physiol Heart Circ Physiol284:H876-H883, 2003, the disclosure of which is incorporated herein byreference. Briefly, approximately 100 microgram protein for Se-16 and 40microgram for Thr-17 were elecrophoresed on 18% SDS-gel, protein wastransferred from the gel to nitrocellulose membrane, the blot was probedwith primary and secondary antibodies and finally bands were visualizedby an ECL method.

It should be noted that tissue homogenate was generally activated atroom temperature, below the normal operating temperature of a heart.This and other features of the results suggest a direct chemical orelectrical effect on the proteins which is possibly divorced or semidivorced from cell function and/or complex biochemical mechanisms (e.g.,more than two or three steps or with feedback). Such divorcing may helpin the application of the effect under various conditions includingvarious polarization conditions and tissue health states. In anexemplary embodiment of the invention, it is noted that the effect doesnot directly depend on the membrane polarization of the cell, therefore,this phosphorylation effect may be achieved at times other than arefractory period. Depending on the non-excitatory signal applied,therapy may be applied during a refractory period to avoid inadvertentpacing. However, if the tissue is desensitized, for example, using asuitable electrical signal, cold, or pharmaceutical, there is no needfor specific timing. In another example, suitable charge provided duringa pacing signal may be sufficient for a therapeutic effect.

For example, a typical pacing pulse is up to 1 ms and 5V, which at 500Ohm lead impedance is 50 micro-joule. A CCM pulse as described herein is2000 micro-joule per pulse and more if multiple pulses are provided in asingle sequence (e.g., 4 pulses=8000 micro-joules).

In an exemplary embodiment of the invention, the provided charge perbeat is at least 100, at least 300, at least 500, at least 1000, atleast 2000, at least 5000 or more or intermediate values ofmicro-joules. It is hypothesized, that at least for some treatments, theapplied field has to be above a minimum charge per heart beat, or theeffect is lost, for example, due to electrical masking in the cell ordue to biochemical interactions that occur within a heart beat.

In an exemplary embodiment of the invention, the applied energy isdirected mostly at a tissue having a volume of less than 20 cm³, lessthan 10 cm³, less than 5 cm³, less than 3 cm³ or larger or intermediatevolumes.

Phosphorylation Dependency on Protein Kinese (PK) Inhibitors

Additional experiments of tissue homogenate were carried out in thepresence and absence of protein kinese inhibitors. The homogenate wasthe same as above, from dogs with heart failure. Two differentphosphorylation locations (threonin-17 and serine-16) on phospholambanproduced two different results in the presence of the kinese inhibitors(in its absence, the above results were reproduced). The kineseinhibitor is STAUROSPORIN, a Pale yellow solid. Advertised as a potent,cell-permeable, and broad spectrum inhibitor of protein kinases.Inhibits protein kinase A (IC₅₀=7 nM), CaM kinase (IC₅₀=20 nM), myosinlight chain kinase (IC₅₀=1.3 nM), protein kinase C (IC₅₀=700 pM), andprotein kinase G (IC₅₀=8.5 nM). Also inhibits platelet aggregationinduced by collagen or ADP but has no effect on thrombin-inducedplatelet aggregation. Induces apoptosis in human malignant glioma celllines. Arrests normal cells at the G₁ checkpoint. Purity: ≧97% by HPLC.CAS 62996-74-1. In the presence of the kinese inhibitors serine-16responded close to usual, possibly with a somewhat delayed and/orreduced response (statistics are weak) and threonin-17 has a reduced anddelayed response, in that no immediate effect was apparent, but someeffect was apparent under longer stimulation times. This suggests twothings. First, the pathways for phosphorylation are different for thetwo locations, when stimulated by an electric field. Second, thereappears to be a synergistic effect between kinese and electric fieldapplication. These differential and synergist effects suggest theability to select pathways in affecting tissue

Without being limited to any particular hypothesis, it is hypothesizedby the inventors that the synergistic effect is caused by the behaviorof Akt (or similar proteins), which is described in a paper Gallo P,Santonastasi M, Ceci M, Scimia C, Di Sciascio G, Condorelli G. Aktoverexpression improves cardiac function and ameliorates heart failurein pressure overload animal model phosphorylating key Ca2+ handlingproteins. J Am Coll Cardiol 2006; 21:76 A, the disclosure of which isincorporated herein by reference. In this paper, it is shown that Aktselectively phosphorylates phospholamban at the threonin-17 site. Inthis, non-limiting explanation, the different sites on phospholamban arephysiologically set up to be phosphorylated under different mechanismand while an electric field has a direct effect on both locations, onelocation is more sensitive to electric fields and the other location ismore sensitive to biochemical interaction (or at least such as mediatedby kinese). It is further hypothesized that the effect of the electricfield applied by the therapy mimics (possibly in an enhanced manner) aregulated effect provided naturally by electrical activity of the heart.

The following tables summarize the experimental results for serine-16:

Control Control + CCM Control + PK Inhibitor PK Inhibitor + CCM P-PLBwith CCM Signals Delivered for 10 sec  5.9 19.9  8.0 22.9  7.6 19.3  9.015.1 13.5 21.2 13.7 11.0 Av  9.0 20.2 10.2 16.3 P-PLB with CCM SignalsDelivered for 20 sec  8.6 26.7 14.0 14.1  6.9 15.6 12.2 18.2 10.4 16.910.6 17.5 Av  8.6 19.7 12.3 16.6 P-PLB with CCM Signals Delivered for 30sec  8.8 21.5  6.8 14.3 15.0 20.2  9.7 18.1 18.0 11.1 11.9 13.0 Av 13.917.6  9.4 15.1 P-PLB with CCM Signals Delivered for 60 sec 12.5 17.515.0 22.7 10.3 17.1  9.8 17.1  7.2 24.3 Av 10.0 19.6 12.4 19.9

The following tables summarize the experimental results for theronin-17:

Control Control + CCM Control + PK Inhibitor PK Inhibitor + CCM P-PLBwith CCM Signals Delivered for 10 sec  7.8 10.2  8.9 14.6 10.2 11.4 12.2 9.9 10.7  7.3  9.5  8.7 Av  9.5  9.6 10.2 11.1 P-PLB with CCM SignalsDelivered for 20 sec  8.5 26.4 13.2 15.0  9.1 41.1  6.2 12.5 16.0 42.3 7.6  8.3 Av 11.2 36.6  9.0 12.0 P-PLB with CCM Signals Delivered for 30sec  7.4 16.1  8.2 24.0  5.4 13.4 12.8 11.5  7.7  9.5 16.0 14.9 Av  6.813.0 12.3 16.8 P-PLB with CCM Signals Delivered for 60 sec  8.7 28.018.3 17.1 13.2 20.5 12.1 12.9 15.2 17.8 11.2 Av 12.4 22.1 13.9 15.0Failed Cardiomyocytes, Pacing and CCM

FIG. 3C shows phosphorylation of a Ryanodine receptor in isolated(in-vitro) failed cardiomyocytes, after application of CCM for 10, 20,30 and 60 seconds. Lack of significant immediate effect is consistentwith the lack of long-term effect shown above and serves to show thatthe effect of the CCM signal can be made specific to certain proteins.

FIG. 3D shows phosphorylation of PLB and GATA, as compared to that ofCSQ, in failed isolated myocytes. As can be seen a phosphorylationeffect is shown for some of the proteins, which matches the generalresults for tissue homogenate. Also noteworthy is that the effectincreases over time at different rates for different proteins.Relaxation times of phosphorylation levels for different proteins arealso generally different. The change in GATA-4 is important because whenGATA-4 is phosphorylated, this process decreases the activity of thesodium calcium exchanger and helps contractility improve quickly.

FIG. 3E shows that a pacing signal applied at 3V for pulses of 0.75 msecdid not have any significant immediate effect on any of the proteins.Possibly, this is caused by the reduced current density of the pacingpulse and/or due to the substantially lower charge transport rate.Possibly, any minimal effect of the pacing signal is relaxed in thetimes between signals. Optionally, the pacing signal is not strongenough to pass a certain minimal threshold of effect.

In Vivo Heart

The following tables summarize the results from 2 heart failure dogs inwhich phosphorylation of phospholamban (PLB-P) after application of CCMsignals was studied, while taking biopsies at 1, 5, 10, and 15 minutes.Normalization to CSQ was used to correct for any effect of blood in thebiopsies. The increase in PLB-P matches a measured increase in dP/dtmeasured in these experiments. It is noted that PLB-P levels remainedelevated for 15 minutes after application of CCM, suggesting atemporally sparse field application treatment. Optionally, such aprolonged elevation has a long term effect on mRNA expression. Variouslocations on the LV were tried, all with similar results, as shownbelow.

PLB-P @ PLB-P @ CCM Time CSQ Ser-16 Thr-17 PLB-P Normalized to CSQ (min)(du) (du) (du) Ser-16 Thr-17 First dog 0 146.09 34.80 28.98 0.24 0.20First set 1 145.94 60.61 34.72 0.42 0.24 5 125.88 60.67 50.51 0.48 0.4010 103.76 92.46 33.09 0.89 0.32 First dog 0 108.38 21.94 25.55 0.20 0.24Second set 1 107.27 71.44 82.46 0.67 0.77 5 112.83 63.31 61.28 0.56 0.5410 72.57 32.85 61.98 0.45 0.85 First dog 0 103.06 26.76 20.16 0.26 0.20Third set 1 116.54 65.60 96.83 0.56 0.83 5 139.30 79.67 144.88 0.57 1.0410 112.53 61.41 62.99 0.55 0.56 15 121.23 55.86 68.86 0.46 0.57 SecondDog 0 117.25 20.93 16.76 0.18 0.14 First set 1 119.63 36.03 33.77 0.300.28 5 132.55 39.08 38.74 0.29 0.29 10 80.44 22.24 16.80 0.28 0.21Second Dog 0 111.84 33.72 40.98 0.30 0.37 Second set 1 118.21 62.7171.28 0.53 0.60 5 68.39 31.31 32.52 0.46 0.48 10 38.79 26.91 29.31 0.690.76 Second Dog 0 114.50 24.17 28.37 0.21 0.25 Third set 1 121.82 44.5262.01 0.37 0.51 5 122.01 46.91 68.04 0.38 0.56 10 134.98 69.64 94.130.52 0.70 15 86.65 35.72 31.55 0.41 0.36

Average of all 6 sets obtained from 2 dogs CCM PLB-P Normalized Time toCSQ (min) Ser-16 Thr-17 0 0.23 0.23 1 0.47 0.54 5 0.46 0.55 10 0.56 0.5715 0.44 0.47 Two sets only (3rd set from each dog)

Without being limited to any particular hypothesis, it is hypothesizedby the inventors that the applied electric field either has a directeffect on the proteins or has an effect on a cofactor or protein thatenhance phosphorylation of proteins. The above “Voltage-dependentpotentiation . . . ” paper suggests that an electric field can directlymodify the natural phosphorylation rate of a protein.

Human Results

mRNA expression levels were measured for some genes in human subjects.Therapy with non-excitatory cardiac contractility modulation (CCM)electrical signals was delivered to LV muscle during the absoluterefractory period improves LV function in patients with HF. The effectsof 3 months CCM therapy on mRNA expression of cardiac fetal and SR genesin 5 patients with advanced HF were examined. In the experiment, rightsided endomyocardial biopsies were obtained at baseline, prior toactivating CCM therapy, and at 3 and 6 months thereafter. CCM therapywas delivered in random order of ON for 3 months and OFF for 3 months.mRNA expression measurement was performed in a blinded fashion as to theON/OFF order of therapy. Expression of the fetal genes A-type (ANP) andB-type (BNP) natriuretic peptides and α-myosin heavy chain (MHC), andthe SR genes SERCA-2a, phospholamban (PLB) and ryanodine receptors (RYR)was measured using RT-PCR and bands quantified in densitometric units(du). The percent change in du between baseline and the ON and OFF 3months phases was calculated.

The 3 months therapy OFF phase was associated with increased expressionof ANP and BNP and decreased expression of α-MHC, SERCA-2a, PLB and RYR(Table). In contrast, the 3 months ON therapy phase resulted indecreased expression of ANP and BNP and increased expression of α-MHC,SERCA-2a, PLB and RYR (Table). This suggests that in patients with HF,CCM therapy reverses the cardiac maladaptive fetal gene program andnormalizes expression of key SR Ca2+ cycling genes. These observationsare consistent with the observed improvement in LV function in patientswith HF following long-term CCM therapy.

mRNA Expression (% Change from Baseline) OFF Phase ON Phase P-Value ANP82 ± 26 −57 ± 9  0.009 BNP 81 ± 28 −55 ± 9  0.007 α-MHC −29 ± 9      80± 16 0.004 SERCA-2a −21 ± 10     45 ± 14 0.039 PLB  4 ± 18   93 ± 450.084 RYR −20 ± 6      34 ± 6  0.002Protein Results

FIGS. 5A-5R shows protein expression results for the following proteinsin chronic dogs, in control, heart failure and treated heart failureconditions: CSQ, SERCA-2a, PLB, RyR, NCX, IL-6, GATA-4, GAPDH, MMP-9,Tubulin-Beta, GATA-1, MMP-1, Tubulin-Alpha, Titin, TIMP-1, Integrin-α5,TNF α, p21ras, p38 MAPK, TIMP-2, β₁-AR, MMP-2, ANP and BNP.

It should be noted that some blots are shown twice, in order tofacilitate comparison between them.

Following is a tabular analysis of these results with a shortdiscussion.

FIGS. 5A-5D show results for the following SR proteins: Calsequestrin,phospholamban, SERCA-2a (Calcium ATPase) and ryanodine receptors; thefollowing Pump Proteins: Sodium-Calcium Exchanger; the followingTranscription Factors: GATA-4; and the following Cytokines:Interleukin-6.

In general, these seven proteins moved directionally the same as theirmRNA expression. Phospholamban showed complete normalization as didSERCA-2a.

These results are consistent with the concept that the CCM acute andchronic effect is mediated by favorable modification of calcium cyclingwithin the sarcoplasmic reticulum.

Also notable is that under chronic condition, the CCM signals appear tonormalize the over-expression of the sodium-calcium exchanger.

Re FIG. 5A:

Dog Numbers - same for all FIGS. 5A-5R HF-Control HF + CCM 02-097 02-10602-098 02-107 02-103 02-108 02-130 02-012 03-045 03-023 04-004 03-05004-018 04-005

Protein Expression of CSQ NL HF-Control HF + CCM 49 41 33 41 44 40 38 3348 45 42 38 50 41 40 34 37 47 44 61 Mean 43 40 44 STD 6 4 9 SEM 3 1 3ANOVA = 0.49 p vs. NL p vs. HF-Control

Protein Expression of Phospholamban NL HF-Control HF + CCM 64 30 75 6323 60 74 27 97 63 29 65 75 18 80 52 30 76 29 69 Mean 65 27 75 STD 8 5 12SEM 3 2 5 ANOVA = 0.0001 p vs. NL <0.05 NS p vs. HF-Control <0.05

Protein Expression of SERCA-2a NL HF-Control HF + CCM 77 37 94 77 69 6789 54 68 117 58 57 95 39 59 74 57 111 63 53 88 54 73 16 12 21 7 4 8ANOVA = 0.007 p vs. NL <0.05 NS p vs. HF-Control <0.05

Protein Expression of RyR NL HF-Control HF + CCM 129 75 115 123 98 107153 102 119 104 86 86 140 100 78 104 86 91 72 147 126 88 106 20 12 23 85 9 ANOVA = 0.009 p vs. NL <0.05 NS p vs. HF-Control NSRe FIG. 5B:

Protein Expression of NCX NL HF-Control HF + CCM 26 62 30 43 50 39 29 3835 36 45 29 41 49 50 44 76 25 58 51 37 54 37 8 12 10 3 5 4 ANOVA = 0.013p vs. NL <0.05 NS p vs. HF-Control <0.05Re FIG. 5C:

Protein Expression of IL-6 NL HF-Control HF + CCM 56 74 31 56 51 70 4387 51 50 84 106 47 107 65 86 118 66 90 51 56 87 63 15 22 23 6 8 9 ANOVA= 0.033 p vs. NL <0.05 NS p vs. HF-Control <0.05Re FIG. 5D:

Protein Expression of GATA-4 NL HF-Control HF + CCM 90 158 63 97 179 9375 100 129 105 133 126 103 157 113 106 127 103 110 141 96 138 110 12 2826 5 11 10 ANOVA = 0.018 p vs. NL <0.05 NS p vs. HF-Control <0.05

FIGS. 5E-5H show results for GAPDH (Housekeeping), transcription factorGATA-1 which did not change, matrix metalloproteinase-9 which changesconsistent with mRNA expression and cytoskeletal protein Tubulin-betawhich also changes consistent with what is shown for mRNA expression.GATA-1 is shown in comparison with GATA-4.

Re FIG. 5E

Protein Expression of GAPDH NL HF-Control HF + CCM 32 34 34 36 33 36 3537 32 35 32 28 34 31 35 31 27 46 27 30 34 32 34 2 4 6 1 1 2 ANOVA = 0.54p vs. NL NS NS p vs. HF-Control NSRe FIG. 5F:

Protein Expression of MMP-9 NL HF-Control HF + CCM 394 637 668 448 779569 452 821 611 455 733 551 488 687 504 426 643 572 742 486 444 720 56632 69 62 13 26 23 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control<0.05Re FIG. 5G:

Tubulin-beta NL HF-Control HF + CCM 512 1008 656 644 810 693 584 973 647450 725 543 390 693 654 301 778 675 1078 689 480 866 651 126 151 51 5257 19 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control <0.05Re FIG. 5H

Protein Expression of GATA-1 NL HF-Control HF + CCM 83 108 118 97 110121 99 60 130 115 109 122 106 106 121 93 94 75 103 101 99 99 113 11 1819 4 7 7 ANOVA = 0.22 p vs. NL NS NS p vs. HF-Control NS

FIGS. 5I-5L show results from the proteins matrix-metalloproteinase-1(MMP-1), cytoskeletal proteins tubulin alpha and titin, tissue inhibitorof matrix-metalloproteinase-1 (TIMP-1) and cell surface proteinintegrin-alpha-5.

There were no apparent changes in TIMP-1. CCM therapy also had nosignificant effect on integrin-alpha-5. It should be noted thatintegrin-alpha-5 can be affected by other means, such as mechanicallyconstraining the heart (e.g., thus directly affecting its transductionfunction).

CCM therapy, however, significantly down-regulated MMP-1, tubulin-alphaand titin which is consistent with the observation with respect to theeffects of CCM on mRNA expression of these genes.

Re FIG. 5I

Protein Expression of MMP-1 NL HF-Control HF + CCM 521 936 482 449 894574 425 883 511 484 1066 538 437 985 527 525 997 539 971 476 474 962 52143 63 34 18 24 13 ANOVA = 0.0001 p vs. NL <0.05 NS p vs. HF- Control<0.05Re FIG. 5J

Tubulin-alpha NL HF-Control HF + CCM 196 298 217 161 322 233 129 359 187136 283 214 142 307 239 158 260 257 274 250 154 300 228 24 33 24 10 13 9ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control <0.05

Protein Expression of Titin NL HF-Control HF + CCM 609 232 368 575 211285 528 218 306 412 302 213 400 231 248 467 223 329 191 243 499 230 28586 35 54 35 13 20 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-ControlNSRe FIG. 5K:

Protein Expression of TIMP-1 NL HF-Control HF + CCM 609 914 786 718 835849 915 863 811 694 679 888 724 773 781 834 676 762 787 709 749 790 798109 90 58 44 34 22 ANOVA = 0.57 p vs. NL NS NS p vs. HF-Control NSRe FIG. 5L:

Integrin-alpha-5 NL HF-Control HF + CCM 228 340 254 153 455 239 160 437212 223 358 193 185 332 168 201 356 253 324 249 192 372 224 31 52 34 1320 13 ANOVA = 0.0001 p vs. NL <0.05 NS p vs. HF-Control <0.05

FIGS. 5M-5P show results from the proteins TNF-α (showed in comparisonto IL-6), p21ras, p38 MAPK, TIMP-2 (showed in comparison to TIMP-1 andβ1-AR.

The lack of change in TIMP-2 is consistent with previous observations.Long-term CCM therapy significantly reduced protein expression of thecytokine TNF-α and significantly reduced the expression of the stretchproteins p21ras as well as p38 MAPK. This is consistent with theobservation that CCM therapy attenuates cardiomyocyte hypertrophy. Alsoto be noted is up-regulation of the beta-1 adrenergic receptor, which isfavorable.

Re FIG. 5M

Protein Expression of IL-6 NL HF-Control HF + CCM 56 74 31 56 51 70 4387 51 50 84 106 47 107 65 86 118 66 90 51 56 87 63 15 22 23 6 8 9 ANOVA= 0.033 p vs. NL <0.05 NS p vs. HF-Control <0.05

Protein Expression of TNFα NL HF-Control HF + CCM 44 155 63 44 161 89 31149 53 47 125 87 51 168 75 39 180 96 176 80 43 159 78 7 19 15 3 7 6ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control <0.05Re FIG. 5N:

Protein Expression of p21ras NL HF-Control HF + CCM 44 112 92 57 142 8938 138 83 30 123 110 34 66 61 36 73 53 90 72 40 106 80 10 31 20 4 12 7ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control <0.05

Protein Expression of p38 MAPK NL HF-Control HF + CCM 21 46 32 15 52 2113 41 35 17 67 36 14 43 24 19 38 19 33 15 17 46 26 3 11 8 1 4 3 ANOVA =0.0001 p vs. NL <0.05 NS p vs. HF-Control <0.05Re FIG. 5O:

Protein Expression of TIMP-2 NL HF-Control HF + CCM 84 62 85 73 55 80 6143 68 93 89 72 87 88 56 88 84 79 81 86 81 72 75 12 18 11 5 7 4 ANOVA =0.53 p vs. NL NS NS p vs. HF-Control NSRe FIG. 5P:

Protein Expression of β1-AR NL HF-Control HF + CCM 135 66 86 75 30 97 8841 53 111 64 95 111 113 86 93 56 96 64 113 102 62 89 21 26 18 9 10 7ANOVA = 0.015 p vs. NL <0.05 NS p vs. HF-Control <0.05

FIGS. 5Q and 5R show results for MMP-2 (in comparison to MMP-1 andMMP-9) and ANP and BNP.

Re FIG. 5Q:

Protein Expression of MMP-2 NL HF-Control HF + CCM 47 74 41 29 77 30 3157 25 42 78 56 38 81 63 42 51 65 41 61 38 66 49 7 16 16 3 6 6 ANOVA =0.0001 p vs. NL <0.05 NS p vs. HF-Control <0.05Re FIG. 5R:

Protein Expression of ANP NL HF-Control HF + CCM 64 104 62 77 179 60 108271 39 82 128 89 92 135 62 103 113 76 97 54 88 147 63 17 61 16 7 23 6ANOVA = 0.002 p vs. NL <0.05 NS p vs. HF-Control <0.05

Protein Expression of BNP NL HF-Control HF + CCM 31 84 78 62 94 62 107111 69 65 87 69 57 94 84 66 77 77 89 73 65 91 73 25 11 7 10 4 3 ANOVA =0.002 p vs. NL <0.05 NS p vs. HF-Control <0.05Comparison of mRNA Levels and Protein Levels in the LV Free Wall onChronic Treated Dogs

Referring back to the 14 dogs tested for chronic effects, mRNAexpression in LV free wall of the housekeeping gene GAPDH and CSQ, thefetal program genes consisting of β₁-AR, αMHC, ANP, and BNP and thecardiac SR genes SERCA-2a, PLB, and RyR are shown in table 5 quantifiedin densitometric units. Expression of GAPDH and CSQ was unchanged amongthe 3 study groups namely, normal dogs, sham-operated HF dogs and HFCMM-treated dogs. mRNA expression of β₁-AR, αMHC, SERCA-2a, PLB and RyRdecreased and expression of ANP and BNP increased significantly insham-operated HF dogs compared to normal. Three months of CCM therapyrestored the expression of all genes to near normal levels. Proteinexpression in the LV free wall of CSQ, β₁-AR, ANP, BNP SERCA-2a, PLB andRyR are also shown in Table 5 quantified in densitometric units. Proteinlevels of CSQ were unchanged among the 3 study groups. Protein levels ofβ₁-AR, SERCA-2a, PLB and RyR decreased and that of ANP and BNP increasedsignificantly in sham-operated HF dogs compared to normal dogs.Long-term CCM therapy restored the expression of all measured proteinsexcept PLB to near normal levels (Table 5). Possibly, phospholamban wasnot restored because of the differential phosphorylation thereof. In anexemplary embodiment of the invention, this mechanism is used toselectively increase synthesis of some proteins over others.

In an exemplary embodiment of the invention, this mechanism is used totest if a patient is getting better—by stopping a therapy which isdirectly causing phosphohorylation and seeing if phospholamban levelsnormalize (or trend to) after a few days and/or if other protein levelstrend towards disease state values.

In an exemplary embodiment of the invention, relaxation time betweensignal application is an integral part of therapy. In one example,relaxation time is used to allow the cell to find a new balance betweenthe expressed proteins that are not on corresponding levels, such abalance may include protein levels (or mRNA levels or phosphorylation orstructural remodeling) degrading and/or protein levels improving. Suchrelation times may be on order of seconds, minutes hours or days,depending on which mechanism are to be allowed to take part (e.g.,protein based, etc.).

In an exemplary embodiment of the invention, when applyingphosphorylation-modifying therapy as described herein, a process ofweaning is applied. Possibly, if treatment is stopped suddenly,phospholamban levels will be too low to support suitable cardiacactivity, possibly causing a downwards-spiral in patient health. In anexemplary embodiment of the invention, as protein levels normalize (forother proteins), therapy is reduced to allow phospholamban recovery.Optionally, the pauses are timed according to measured recovery inphospholamban levels.

In an exemplary embodiment of the invention, electrical therapy isapplied selectively to tissue measured as having reduced phospholambanlevels and/or phosphorylation levels, so as not to potentially damagehealthy tissue. In some cases, changes to “healthy” tissue is desirable.For example, increasing phosphorylation and thus possibly reducingphospholamban may be desirable if long term reduction in contractilityis desired. In another example, phosphorylation may be increased innormal tissue in order to cause over (or under) expression of someproteins, such as gap junction proteins or mechanical proteins. Itshould be appreciated that a therapy target of diseased tissue need notbe a mirror of a healthy tissue state.

TABLE 5 mRNA and Protein Expression of Fetal Program and SarcoplasmicReticulum Genes/Proteins in Left Ventricular Free Wall of Normal Dogs(NL) (n = 6), Sham-Operated Untreated Heart Failure Dogs (Sham, n = 7)and CCM-Treated Heart Failure Dogs (CCM, n = 7) mRNA Expression (du)Protein Expression (du) NL Sham CCM NL Sham CCM B₁-AR  22 ± 1 11 ± 1* 18 ± 1*† 102 ± 9   62 ± 10* 89 ± 7† A-MHC 212 ± 7 148 ± 10* 180 ± 5*† —— — ANP  18 ± 2 34 ± 2*  27 ± 1*† 88 ± 7 147 ± 23* 63 ± 6† BNP  23 ± 3177 ± 4*   44 ± 5*†  65 ± 10 91 ± 4* 73 ± 3† SERCA-2a 236 ± 4 154 ± 4* 192 ± 3*† 88 ± 7 54 ± 4* 73 ± 8† PLB 232 ± 8 149 ± 8*  212 ± 7†  446 ±19 277 ± 12* 299 ± 9*  RyR  38 ± 2 22 ± 2* 36 ± 5† 126 ± 8  88 ± 5* 106± 9†  CSQ 264 ± 5 275 ± 5  259 ± 14  43 ± 3 40 ± 1  44 ± 3  GAPDH 219 ±6 223 ± 8  236 ± 4  — — — du = Densitometric units; AR = Adrenergicreceptor; MHC = Myosin Heavy Chain; ANP = A-type natriuretic peptide;BNP = B-type natriuretic peptide; SERCA-2a = Cardiac sarcoplasmicreticulum calcium ATPase; PLB = Phospholamban; RyR = Ryanodine receptor;CSQ = Calsequestrin; GAPDH = Glyseraldehyde-3-phosphate dehydrogenase.*= p < 0.05 vs. NL; †= p < 0.05 vs. Sham.Local and Remote Effects

The above results showed analysis of tissue samples at the treated site.FIG. 6 shows mRNA expression levels for Phospholamban, SERCA-2a andRyanodine receptors, showing chronic improvement in septal tissue towhich a field was applied chronically.

Referring to the 14 dogs chronic study described above, the restorationto near normal levels of the fetal program and most SR proteins after 3months of CCM therapy was the same in LV tissue obtained from theinter-ventricular septum, the site nearest to the CCM signal deliveryleads, and the LV free wall, a site remote from the CCM leads. A typicalexample illustrating the changes in protein levels of CSQ, SERCA-2a, PLBand RyR between the two LV sites is shown in FIG. 10.

FIG. 8 shows phosphorylation levels in chronically treated dogs, at theapplication location in the septum.

In addition to examining protein expression of fetal program genes andSR proteins, protein levels of P-PLB at serine-16 and threonine-17 werealso examined. Measurements were made in tissue obtained from both theinter-ventricular septum and the LV free wall. At both sites, proteinlevels of P-PLB at serine-16 and threonine-17 were significantly lowerin sham-operated HF dogs compared to normal dogs and returned to nearnormal levels after 3 months of CCM therapy (FIG. 11, Table 6). In boththe inter-ventricular septum and LV free wall, the ratio of P-PLB atserine-16 to total PLB and the ratio of P-PLB at threnonine-17 were alsosignificantly lower in sham-operated HF dogs compared to normal dogs(Table 6). Long-term CCM therapy resulted in a significant increase ofboth ratios in both the interventricular Septum and the LV free wall(Table 6).

TABLE 6 Protein Expression of Total Phospholamban and PhosphorylatedPhospholamban at Serine-16 and Threonine-17 in the Inter-VentricularSeptum and Left Ventricle Free Wall of Normal Dogs (NL) (n = 6), Sham-Operated Untreated Heart Failure Dogs (Sham, n = 7) and CCM-TreatedHeart Failure Dogs (CCM, n = 7)) Inter-Ventricular Septum LV Free WallNL Sham CCM NL Sham CCM Total PLB (du) 445 ± 7  305 ± 23*  299 ± 16* 446± 19  305 ± 19*  299 ± 9*  P-PLB Ser-16 (du) 85 ± 6  47 ± 5*  79 ± 6†128 ± 11  50 ± 12*  87 ± 11*† P-PLB Thr-17 (du) 146 ± 6  62 ± 10* 129 ±12† 137 ± 4  56 ± 7*  109 ± 18†  P-PLB Ser-16/Total PLB 0.19 ± 0.01 0.15± 0.01*  0.27 ± 0.02† 0.29 ± 0.03 0.16 ± 0.03* 0.29 ± 0.04† P-PLBThr-17/Total PLB 0.33 ± 0.01 0.21 ± 0.04*  0.44 ± 0.05† 0.31 ± 0.02 0.19± 0.03* 0.36 ± 0.05† PLB = phospholamban; P-PLB = phosphorylatedphospholamban; Ser-16 = serine-16; Thr-17 = theonine-17. *= p < 0.05 vs.NL; †= p < 0.05 vs. Sham.

FIGS. 7A and 7B shows mRNA expression at sites remote from theapplication of the signal, but still within the left ventricle, at arelatively short time of four hours apparently no significant effect(mRNA, protein and/or phosphorylation) is shown. This may indicate thatthe effect of the CCM signal is first local, for example on a molecularlevel and then propagates to remote location, for example by biochemicalsignaling or by a mechanical signaling indicated by the change incontraction behavior of the treated tissue and/or of the chamber as awhole. The following non-limiting mechanism is suggested: the electricfield causes phosphorylation of phospholamban. This in turn increasesthe activity/affinity of SRECA-2a for calcium and immediately improvesSR calcium cycling. GATA-4 and the sodium calcium exchanger may play anadditive role in the improved function. As LV function begins to improveand the LV gets smaller, many of the molecular/biochemicalmaladaptations begin to correct, which adds to the long-term benefits.Improved SR cardiac cycling may be a goal of some therapies inaccordance with exemplary embodiments of the invention.

In open chest HF dogs, acute hemodynamic and local/remote effects weredetermined. Continuous delivery of CCM therapy over the course of 2hours improved LV systolic function and associated hemodynamics.Compared to baseline before initiating CCM therapy, 2 hours of CCMtherapy resulted in a significant increase of LV ejection fraction (31±2vs. 26±1%, p<0.05), a reduction of MVO₂ that did not reach statisticalsignificance (180±34 vs. 257±41 μmols/min) and a trend toward anincrease in LV mechanical efficiency that also did not reach statisticalsignificance (33±8 vs. 19±4%).

LV tissue obtained near the site of CCM lead implants, was compared toLV tissue obtained from a site remote from the CCM leads. LV tissuesamples obtained from the same sites from dogs with HF that wereuntreated and normal dogs were used for comparisons. Compared tountreated HF dogs, the ratio of P-PLB to total PLB increasedsignificantly in CCM-treated HF dogs compared to untreated HF dogs inthe LV anterior wall at the site of signal delivery (FIG. 12A), whereasit was essentially unchanged in the LV posterior wall remote from thesite of CCM signal delivery (FIG. 12B).

In an exemplary embodiment of the invention, the location to whichelectrification will be applied is selected based on a model of whatareas will cause a biochemical or mechanical cascade in the heart andimprove its function. Optionally, the size of areas is optimized toreduce power needs, while retain an expected time frame of treatment.

In an exemplary embodiment of the invention, an area to be treated isselected based on immediate response of tissue therein to electricalstimulation.

One example of a mechanical cascade is a desired change in stretching oftissue which will, as that tissue improve, propagate. Another example ofmechanical cascade is selecting sufficient and correct tissue in theventricle such that immediate hemodynamic effects (e.g., improvement)are seen and sensed by the rest of the chamber.

A possible mechanism of non-mechanical propagation (which may beutilized) is that healthy cells can help regulate ionic concentrationsof neighboring cells via gap junctions between them. Alternatively oradditionally, other communication may occur via the gap junctions.

Another possible mechanism is that low level fields reach further andtake longer to have an effect. This suggests that low-level fields ingeneral may be used, with a rapidity of effect depending on the fieldstrength and/or mechanical or other improvement of parts of the heart.

In some embodiments of the invention, the efficacy of a treatment ismeasured by tracking remote effects alternatively or additionally, totracking local effects. One or more of the following logics may beused: 1. There is more remote tissue to sample, with less danger ofdamage to heart. 2. The effect in remote tissue is more gradual and thuseasier to track. 3. Acute effects may not occur (or be smaller) inremote tissue, thereby preventing the masking of longest term effects byacute effects. Optionally, for local or remote measurement, themeasurement is made when no treatment is applied. An example of an acuteeffect which may mask a longer term effect is conduction velocity.

A short tabular summary of the results of FIGS. 6-8 follows:

Re FIG. 6

Dog Numbers HF-Control HF + CCM 02-097 02-106 02-098 02-107 02-10302-108 02-130 02-012 03-045 03-023 04-004 03-050 04-018 04-005

mRNA Expression for SERCA-2a in Septum NL HF-Control HF + CCM 238 172207 224 123 214 203 167 178 199 168 191 226 151 179 162 123 150 149 162Mean 209 150 183 STD 27 21 23 SEM 11 8 9 ANOVA = 0.001 p vs. NL <0.05 NSp vs. HF-Control <0.05

mRNA Expression for PLB in Septum NL HF-Control HF + CCM 241 255 394 330277 374 324 281 327 344 276 364 368 290 352 352 275 292 278 243 Mean 327276 335 STD 45 11 52 SEM 18 4 20 ANOVA = 0.029 p vs. NL <0.05 NS p vs.HF-Control <0.05

mRNA Expression for RYR in Septum NL HF-Control HF + CCM 272 287 267 371225 277 289 229 300 294 262 319 321 232 323 294 234 252 238 248 307 244284 35 23 31 14 9 12 ANOVA = 0.005 p vs. NL <0.05 NS p vs. HF-Control<0.05Re FIGS. 7A and 7B:

mRNA Expression for ANP NL HF-Control HF + CCM 155 301 342 164 336 316175 356 307 170 325 303 229 318 291 212 350 282 Mean 184 331 307 STD 2920 21 SEM 12 8 9 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control NS

mRNA Expression for SERCA-2a NL HF-Control HF + CCM 257 68 126 266 50 57334 56 140 293 84 71 285 53 36 263 47 38 Mean 283 60 78 STD 29 14 45 SEM12 6 18 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control NS

mRNA Expression for VEGF NL HF-Control HF + CCM 14 4 8 16 5 6 13 5 7 1914 11 24 8 13 22 9 4 Mean 18 8 8 STD 4 4 3 SEM 2 2 1 ANOVA = 0.001 p vs.NL <0.05 <0.05 p vs. HF-Control NS

mRNA Expressionfor GATA-4 NL HF-Control HF + CCM 121 352 341 129 367 349145 373 333 126 350 325 133 377 300 136 394 262 Mean 132 369 318 STD 816 32 SEM 3 7 13 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control<0.05

mRNA Expression for BNP NL HF-Control HF + CCM 320 1721 1862 299 16292012 331 1690 1952 294 1725 2111 361 1662 1991 349 2016 1641 326 17411928 27 140 162 11 57 66 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs.HF-Control <0.05

mRNA Expression for Phospholamban NL HF- Control HF + CCM 18 9 14 17 911 17 7 11 19 11 9 20 7 6 21 11 4 19 9 9 2 2 4 1 1 1 ANOVA = 0.0001 pvs. NL <0.05 <0.05 p vs. HF-Control NS

mRNA Expression for bFGF NL HF-Control HF + CCM 186 90 110 195 76 100194 108 87 215 77 88 189 104 83 200 100 70 197 93 90 10 14 14 4 6 6ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control NS

mRNA Expression for α-MHC NL HF-Control HF + CCM 457 242 222 609 228 218448 176 208 545 188 221 642 313 181 557 220 156 543 228 201 78 49 27 3220 11 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs. HF-Control NSRe FIG. 8:

Dog Numbers HF-Control HF + CCM 02-097 02-106 02-098 02-107 02-10302-108 02-130 02-012 03-045 03-023 04-004 03-050 04-018 04-005

Phosphorylated PLB at Serine-16 NL HF-Control HF + CCM 143 86 99 109 95106 153 63 68 161 25 97 102 15 105 100 24 31 43 106 Mean 128 50 87 STD28 32 28 SEM 11 12 11 ANOVA = 0.0001 p vs. NL <0.05 <0.05 p vs.HF-Control <0.05

Phosphorylated PLB at Threonine-17 NL HF-Control HF + CCM 135 90 80 12641 68 137 53 62 148 52 162 146 32 168 129 59 78 67 147 137 56 109 9 1947 4 7 18 ANOVA = 0.0001 p vs. NL <0.05 NS p vs. HF-Control <0.05Additional Proteins

Experimental results of the effect of CCM signals on other proteins isdescribed below.

Effect of CCM on Calcium Cycling in the Sarcoplasmic Reticulum and/orS100A1 Protein.

Overexpression of the calcium-binding protein S100A1 in failing ratcardiomyocytes normalizes sarcoplasmic reticulum (SR) calcium cycling byincreasing calcium-uptake and reducing SR calcium-leak from ryanodinechannels. Expression of S100A1 is significantly decreased in leftventricular (LV) myocardium of explanted failed human hearts.

RNA was extracted and homogenate prepared from LV tissue obtained from 6CCM-treated HF dogs, 6 untreated HF dogs and 6 normal (NL) dogs (theseare the same dogs as above). S100A1 mRNA expression was measured usingreverse transcriptase polymerase chain reaction (RT-PCR) and proteinexpression was measured using Western blotting. Bands obtained after gelelectrophoresis were quantified in densitometric units (du).

S100A1 mRNA and protein expression decreased significantly in untreatedHF dogs compared to NL dogs. Chronic CCM therapy significantly increasedmRNA and protein expression of S100A1. Restoration of expression of thiscalcium binding protein improves calcium cycling within the SR and mayaccount, at least in part, for the observed improvement of LV functionseen following chronic CCM therapy.

mRNA and Protein Expression of S100A1

NL HF-Untreated HF + CCM S100A1 mRNA (du) 13.0 ± 0.7  1.5 ± 0.1* 8.6 ±0.5*† S100A1 Protein (du) 215 ± 15 63 ± 6* 190 ± 8†   *= p < 0.05 vs.NL; †= p < 0.05 vs. HF-UntreatedEffect on Sorcin; Presenilin and Calstabin

RNA was extracted and homogenate prepared from LV tissue obtained from 6CCM-treated HF dogs, 6 untreated HF dogs and 6 normal (NL) dogs. Theseare the same dogs as in the previous section. Sorcin, Presenilin andCalstabin mRNA expression was measured using reverse transcriptasepolymerase chain reaction (RT-PCR) and protein expression was measuredusing Western blotting. Bands obtained after gel electrophoresis werequantified in densitometric units (du).

Sorcin mRNA and protein expression decreased significantly in untreatedHF dogs compared to NL dogs. Chronic CCM therapy significantly increasedmRNA and protein expression of Sorcin. Presenilin-2 increased in HF anddecreased with CCM. Preseilin-1 was measured as an internal control andit did not change. This suggests that Presenilin-2 can be used as atarget (e.g., treatment goal) for treating HF and as an indicator fordiagnosis and modification of treatment.

Restoration of expression of Sorcin may prevent or limit the RyR2calcium leak and in doing so improve calcium cycling within the SR.Correction of this maladaptation by CCM therapy may account, at least inpart, for the observed improvement of LV function. In particular, thecombined correction of Sorcin and Presenilin, which interact to regulatecardiac ryanodine calcium release channels may act as a mediator ofrecovery of calcium overload in cardiomyocytes due to “RyR-2 calciumleak” in heart failure.

It should be noted that Calstabin-2 decreased in heart failure butremained depressed even after 3 months of CCM therapy. This suggeststhat CCM does not act by merely resetting cellular function, however, asnoted above, resetting may be part of the process. One possibleexplanation is that Castabin-2 does not rebound as it is tied tophospholamban levels. Another possible explanation is that Castabin-2indicates a path for the progression of heart failure. Until theunderlying cause is removed, it may remain depressed. Possibly, animprovement over significantly longer periods of time is to be expected.In an exemplary embodiment of the invention, this is used to estimatethe ability of a patient to stop therapy. For example, if Castabin-2levels trend to or do normalize, this can indicate that the tissue stateis not diseased or becoming healthier. Optionally, this allows trackingof tissue improvement even during ongoing electrical therapy. In anexemplary embodiment of the invention, signal optimization techniques asdescribed herein are used to find and then apply a signal specific (ormore specific) to modifying Castabin-2.

The results are shown in FIGS. 13-15 and in the tables below (indensitometric units).

FIGS. 13A and 13B present mRNA (FIG. 13A) and protein blots (FIG. 13B)illustrating expression of Sorcin in LV tissue of HF Dogs treated withCCM for 3 months according to an exemplary embodiment of the invention.

FIGS. 14A, 14B, 14C and 14D present mRNA (FIGS. 14A and 14C) and proteinblots (FIGS. 14B and 14D) of the Presenilin-1 (FIGS. 14A and 14B) andPresenilin 2 (FIGS. 14C and 14D) in LV tissue of HF Dogs treated withCCM for 3 months according to an exemplary embodiment of the invention.

FIGS. 15A and 15B present mRNA (FIG. 15A) and protein blots (FIG. 15B)illustrating expression of Calstabin in LV tissue of HF Dogs treatedwith CCM for 3 months according to an exemplary embodiment of theinvention.

Dog Numbers HF-Control HF + CCM 02-097 02-106 02-098 02-107 02-10302-108 02-130 02-012 03-045 03-023 04-018 03-050

mRNA Expression of Sorcin Normal HF-Control HF + CCM 0.52 0.19 0.37 0.410.21 0.37 0.55 0.25 0.30 0.53 0.19 0.29 0.53 0.18 0.39 0.59 0.15 0.37Mean 0.52 0.20 0.35 STD 0.06 0.04 0.04 SEM 0.02 0.02 0.02 ANOVA = 0.0001p vs. Normal <0.05 <0.05 p vs. HF-Control <0.05

Protein Expression of Sorcin Normal HF-Control HF + CCM 0.75 0.30 0.3960.65 0.26 0.456 0.69 0.28 0.426 0.75 0.30 0.412 0.73 0.25 0.426 0.680.23 0.486 Mean 0.71 0.27 0.43 STD 0.04 0.03 0.03 SEM 0.02 0.01 0.01ANOVA = 0.0001 p vs. Normal <0.05 <0.05 p vs. HF-Control <0.05

mRNA Expression of Presenilin-1 Normal HF-Control HF + CCM 0.34 0.4 0.380.36 0.44 0.4 0.38 0.39 0.45 0.39 0.43 0.39 0.29 0.38 0.38 0.32 0.330.35 Mean 0.35 0.40 0.39 STD 0.04 0.04 0.03 SEM 0.02 0.02 0.01 ANOVA =0.131 p vs. Normal NS NS p vs. HF-Control NS

Protein Expression of Presenilin-1 Normal HF-Control HF + CCM 0.12 0.150.14 0.11 0.19 0.13 0.09 0.21 0.14 0.15 0.11 0.16 0.16 0.09 0.18 0.110.13 0.11 Mean 0.12 0.15 0.14 STD 0.03 0.05 0.02 SEM 0.01 0.02 0.01ANOVA = 0.338 p vs. Normal NS NS p vs. HF-Control NS

mRNA Expression of Presenilin-2 Normal HF-Control HF + CCM 0.28 0.630.34 0.32 0.61 0.31 0.29 0.59 0.29 0.35 0.49 0.42 0.2 0.56 0.35 0.310.62 0.38 Mean 0.29 0.58 0.35 STD 0.05 0.05 0.05 SEM 0.02 0.02 0.02ANOVA = 0.0001 p vs. Normal <0.05 NS p vs. HF-Control <0.05

Protein Expression of Presenilin-2 Normal HF-Control HF + CCM 0.201 0.520.25 0.159 0.48 0.29 0.198 0.55 0.21 0.285 0.62 0.19 0.248 0.49 0.260.196 0.61 0.11 Mean 0.21 0.55 0.22 STD 0.04 0.06 0.06 SEM 0.02 0.020.03 ANOVA = 0.0001 p vs. Normal <0.05 NS p vs. HF-Control <0.05

mRNA Expression of Calstabin-2 Normal HF-Control HF + CCM 1667  847 14771488 1204 1355 1486 1389  906 1723  887  619 1778 1120 1497 1840 13431224 Mean 1,664  1,132  1,180  STD  148  227  350 SEM  61  93  143 ANOVA= 0.004 p vs. Normal   <0.05   <0.05 p vs. HF-Control NS

Protein Expression of Calstabin-2 Normal HF-Control HF + CCM 5871 39913679 5602 3643 3923 5029 3156 3560 5579 4645 4083 6726 4567 4600 51904995 4381 Mean 5,666  4,166  4,038  STD  602  693  402 SEM  246  283 164 ANOVA = 0.0001 p vs. Normal   <0.05   <0.05 p vs. HF-Control NSEffect Wash-Out Times

FIGS. 9A and 9B are graphs showing the relationship between rise timeand decay time of a contractility increase in dogs. Similar results wereobserved in humans, albeit with generally slower rise times and slowerdecay times.

FIG. 9A, in which the scale is in seconds, shows a rise time of severaltens of seconds at about time 270. Once the signal is stopped (uppergraph shows the signal application times), the contractility increasedecays. The blip at 600 is probably caused by an arrhythmia. The decaycontinues from 600 until 950. A second signal application is at about950 with a change in signal application location at about time 1350,before the signal effect washed out.

In FIG. 9B the signal is shown as an outline of a square pulse, with thesignal stopped each time an arrhythmia is detected, shown as a shortpause in the signal.

Again, a rise time of several tens of seconds is found. A decay time of230 seconds is shown for the first signal. A longer decay time of over400 seconds is shown for a second signal. The second signal caused anincrease in contractility at about 1050 seconds, due to change in theapplied signals.

In an exemplary embodiment of the invention, it is noted that the acuteand long term effects last at least a few seconds after treatment isstopped and have different wash-out times. Thus, measuring patientparameters is possible while the treatment apparatus is off. Differentwaiting times may be used to selectively measure acute and longer termeffects. For example, 30 minutes may be sufficient for avoiding acuteeffects on phosphorylation.

In an exemplary system, a controller learns the particular washoutbehavior of a patient and adjusts the treatment to be re-applied onlyafter significant washout and/or be applied at a minimal length that hasa washout. Optionally, the delay between application and/or length ofapplications are selected to optimize one or more of power consumption,danger of arrhythmia and/or discomfort to patient.

WO 2005/087310, filed as PCT/IL2005/00316 on Mar. 18, 2005, U.S.provisional application 60/719,517, filed Sep. 22, 2005 and U.S.provisional application 60/654,056, filed February 17th, the disclosuresof which are incorporated herein by reference, describe methods anddevices for treating metabolic diseases. In particular, 60/719,517describes how applying a signal to a stomach has an effect on reducingblood glucose levels which lasts after the signal is stopped, forexample, for more than one day. At least in combination with the resultsdescribed above, this suggests that an electrical therapy may be used tochange the mode of operation of tissue, on a cellular level, possiblyfor tissue in general or for excitable tissue at least.

It is a particular feature of some embodiments of the invention that anon-immediate effect of signal application, which lasts after the signalapplication is stopped, is an affirmative effect, in which the tissueexhibits a positive change in action, rather than a negative effect, forexample, of preventing arrhythmia. In an exemplary embodiment of theinvention, the effect is on a cellular level, rather than or in additionto an effect on an organ level, for example a cellular level as measuredby protein activity and/or expression levels.

Another type of long term/wash-out effect is a long term therapeuticeffect lasting days or weeks after treatment. Below are described twosets of experiments on humans. In the first set, patients wereselectively treated 3 months on and 3 months off or vice versa. In thesecond set of experiments patients were either treated for six months ornot. While the patients that were 3 months on and then 3 off fared lesswell (after 6 months) than those that were 3 off and then 3 on, theoverall situation after 6 months was better than what would be expectedfrom a baseline (no treatment) situation. The second set of experimentswhile suggesting no overall improvement after 6 months, shows a trend tolesser degradation and also suggests what the expected degradation wouldbe. Optionally, the therapies as described herein are used to slow downdegradation of a patient's situation, rather than a treatment.Optionally, each patient is selectively tested to see how long thetreatment effects last and/or what minimal treatment period is requiredfor longer term effects. Optionally, the length of treatment and/or“off” periods between treatments also vary as a function of time.Various physiological measures, for example as described above may beused to assess improvement, as well as mRNA, protein and/orphosphorylation levels.

In the first set of experiments 126 subjects with ischemic (60%) oridiopathic (40%) cardiomyopathy, EF<35%, NYHA Class II (23%) or III(73%) received a CCM pulse generator. Patients were randomly assigned toGroup 1 (n=62, CCM treatment 3 months, sham treatment second 3 months)or Group 2 (n=64, sham treatment 3 months, CCM treatment second 3months). The coprimary endpoints were differences between groups ofchanges in peak oxygen consumption (VO_(2,peak)) and Minnesota Livingwith Heart Failure Questionnaire (MLWHFQ).

Baseline EF (25±6% vs 26±6%), VO_(2,peak) (14.4±2.5 vs 14.2±2.7ml/kg/min) and MLWHFQ (41±20 vs 35±19) were similar in both groups.VO_(2,peak) increased similarly in both groups during the first 3 months(0.64±0.36 vs 0.53±0.46 ml O2/kg/min, p=NS, possibly a placebo effect).However, during the next 3 months, VO_(2,peak) decreased in the groupswitched to sham treatment (−0.92±0.42 ml/kg/min) and continuedincreasing in patients switched to active treatment (0.35±0.35ml/kg/min, p=0.02), so that the final difference between groups was 1.2ml/kg/min. It is hypothesized that this reduction is less than whatwould be expected after 3 months of an untreated patient. MLWHFQ alsoimproved in both groups during the first 3 months (placebo effect),though the improvement trended better with treatment (−14.65±2.04 vs−9.8±2.2, p=NS). During the second 3 months, MLWHFQ increased in thegroup switched to sham (5.1±2.3) and continued to decrease in patientsswitched to active treatment (−1±2, p=0.03). However, the increase inthe untreated group appeared to be less than a return to baseline valuesor other values that would be expected with a diseased patient withnatural progression.

It should be noted that significant improvement is achieved even forpatients that had an ON phase followed by an OFF phase, or at least theeffects of treatment remained for a while. This suggests that thetherapeutic effects are long lasting, for example, more than a week,more than a month, more than three months or longer. This is in contrastto cardiac resynchronization therapy where improvements seem to last aweek or two at most. Even in this case, the heart desynchronizationappears almost immediately (as is the case with electrical therapy ingeneral which usually last a single beat).

In a second set of experiments, 49 subjects with EF<35%, normal QRSduration (105±15 ms) and NYHA Class III (n=48) or IV (n=1) despitemedical therapy received a CCM pulse generator. Devices were programmedrandomly to deliver CCM signals (Treatment, n=25) or to remain off(Control, n=24) for 6 months. Evaluations included NYHA, 6 minute hallwalk (6MW), cardiopulmonary stress test, Minnesota Living with HeartFailure Questionnaire (MLWHFQ) and Halter.

Although most baseline features were balanced between groups, EF(31.4±7.4 vs 24.9±6.5%, p=0.003), end-diastolic dimension (52.1±21.4 vs62.5±6.2 mm, p=0.01), peak VO₂ (16.0±2.9 vs 14.3±2.8 ml O₂/kg/min,p=0.02) and anaerobic threshold (12.3±2.5 vs 10.6±2.4 ml O₂/kg/min,p=0.01) were worse in Treatment than Control. Nevertheless, there was 1death in a Control subject and more Treatment patients were free ofhospitalization for any cause at 6 months (84% vs 62%). No changes inectopy were observed. Compared to baseline, 6 MW (13.4 m), peak VO₂ (0.2ml O₂/kg/min) and anaerobic threshold (0.8 ml O₂/kg/min) increased morein Treatment than Controls. None of the differences were statisticallysignificant (small sample size).

Six minute hall walk showed similar improvements in both groups at 12weeks (˜20 meters), with the curves diverging at 6 months with anapproximately 15 meter greater increase in the treatment group. Peak VO₂decreased over time in both groups (˜−0.75 at 12 W, ˜−1 at 24 weeks),but remained higher in the treatment group than in controls, by ˜0.2 mlO₂/kg/min. In contrast, anaerobic threshold, which decreased in thecontrol group (˜−0.8 at 12 W, 24W), decreased initially (˜−0.6 at 12 W)but then returned to baseline values at 6 months in the Treatment group.At the final follow-up, the difference between the two groups averaged0.82 ml O₂/kg/min. Ejection fraction increased minimally in both groupsat 6 months (1.8±0.8 in the treatment group vs 1.3±1.6 in the controlgroup).

New York Heart Association classification improved similarly in bothgroups. For the treatment group, the proportion of patients in Class I,II and III at 24 weeks were 19, 45 and 36, respectively. This comparedto 18, 52 and 30, respectively, in the Control group. MLWHFQ alsoimproved significantly and similarly in both groups. At the 6 monthfollow-up, MLWHFQ decreased from baseline values by 16.2±5.9 and18.3±4.8 in the control and treatment groups, respectively. Thesignificant and sustained improvements in NYHA and MLWHFQ observed inboth groups speaks to the presence of a significant placebo effect.

Synergistic Interaction of Beta-Blockers and Electrical Therapy

While it might be supposed that beta blockers and electrical therapy asdescribed here utilize similar pathways and therefore cannot beadditive, the following experiment shows that this is not correct. Inaddition, electrical therapy may be used to increase contractility andcompensate for an initial reduction in contractility caused by betablockers. This may suggest changing the sequence amount and/or type whenthe medication starts having a positive effect, for example, severalweeks or several months (e.g., 3) later. Alternatively or additionally,electrical therapy is used to compensate for times when medication isstopped, for example, due to adverse systemic effects. In an exemplaryembodiment of the invention, the dosage of medication and the dosage oftherapy are selected to minimize or reduce systemic effects and/orreduce power needs and/or side effects (if any) of electrical therapy.

In a set of experiments, dogs with intracoronarymicroembolization-induced chronic HF (LV EF 30%-40%) were randomized to3 months therapy with either BB (beta-blockers) alone (extended releasemetoprolol succinate, 100 mg once daily), extended release metoprololsuccinate (100 mg, once daily) combined with CCM therapy, or to notherapy at all (Control). LV end-diastolic volume (EDV), end-systolicvolume (ESV) and EF were measured before randomization (PRE) and after 3months of therapy (POST). To determine “treatment effect”, the change Δbetween PRE and POST therapy for each group was compared among the 3study groups.

In Control dogs, LV EDV and ESV increased significantly and EF decreasedsignificantly. Therapy with BB alone and combined therapy with BB andCCM both prevented the increase in EDV, significantly reduced ESV andsignificantly increased EF. Treatment effect (Δ) comparisons showed thatcombination therapy with BB and CCM significantly increased LV EF abovethat seen with BB alone (12±1% vs. 6±1%, p<0.001).

Control BB Alone BB + CCM PRE POST PRE POST PRE POST LV EDV (ml) 60 ± 164 ± 1* 64 ± 4 62 ± 5  70 ± 4 69 ± 4  LV ESV (ml) 39 ± 1 45 ± 1* 41 ± 236 ± 4* 45 ± 3 36 ± 3* LV EF (%) 36 ± 1 31 ± 1* 36 ± 1 42 ± 2* 36 ± 1 48± 1* *= p < 0.05 PRE vs. POSTFollowing are detailed results:

CCM + Toprol-XL (n = 7) Dog# Base Pre Post EDV (ml) 04-112 61 78 7704-120 51 69 67 04-124 56 72 74 04-145 52 60 59 05-025 51 56 55 05-03351 66 65 05-034 56 64 62 Mean 54 66 66 STD 4 7 8 SEM 1.4 2.8 3.0 ESV(ml) 04-112 29 50 41 04-120 25 44 35 04-124 24 47 40 04-145 22 37 2905-025 24 36 31 05-033 23 41 33 05-034 30 41 32 Mean 25 42 34 STD 3 5 5SEM 1.1 1.9 1.7 EF (%) Dog# Base Pre Post Delta 04-112 52 36 47 1104-120 51 36 48 12 04-124 57 35 46 11 04-145 58 38 51 13 05-025 54 36 448 05-033 55 38 49 11 05-034 46 36 48 12 Mean 53 36 48 11 STD 4 1 2 2 SEM1.5 0.4 0.8 0.6 Topro-XL (n = 6) Dog# Base Pre Post EDV (ml) 03-057 5462 59 03-066 58 74 76 04-147 51 57 55 05-006 55 64 59 05-028 54 60 6005-042 54 59 59 Mean 54 63 61 STD 2 6 7 SEM 0.9 2.5 3.0 ESV (ml) 03-05727 38 33 03-066 26 48 47 04-147 19 38 33 05-006 30 39 32 05-028 30 37 3505-042 25 37 31 Mean 26 40 35 STD 4 4 6 SEM 1.7 1.7 2.4 EF (%) Dog# BasePre Post Delta 03-057 50 38 44 6 03-066 55 35 38 3 04-147 62 33 40 705-006 46 39 46 7 05-028 44 38 42 4 05-042 54 37 47 10 Mean 52 37 43 6STD 7 2 3 2 SEM 2.7 0.9 1.4 1.0 Placebo (n = 6) Dog# Base Pre Post EDV(ml) 03-031 51 59 63 03-065 54 61 68 04-103 52 58 61 04-122 48 63 6405-002 54 61 64 05-027 59 60 64 Mean 53 60 64 STD 4 2 2 SEM 1.5 0.7 0.9ESV (ml) 03-031 24 39 46 03-065 23 39 48 04-103 21 37 43 04-122 23 39 4305-002 28 39 43 05-027 28 39 44 Mean 25 39 45 STD 3 1 2 SEM 1.2 0.3 0.8EF (%) Dog# Base Pre Post Delta 03-031 51 34 27 −7 03-065 48 36 29 −704-103 60 36 30 −6 04-122 52 38 33 −5 05-002 48 36 33 −3 05-027 53 35 31−4 Mean 52 36 31 −5 STD 4 1 2 2 SEM 1.8 0.5 1.0 0.7 EDV = LVend-diastolic volume ESV = LV end-systolic volume EF = LV ejectionfractionDiagnosis

In an exemplary embodiment of the invention, the above experimentalresults are used as a basis for diagnosis. Optionally, the diagnosisindicates one or both of a disease type and disease severity. In anexemplary embodiment of the invention, the type can be seen from thebehavior of the proteins, for example comparing protein levels tocalcium overload to diagnose systolic or diastolic dysfunction. Inanother example, the relative phosphorylation levels are used as ameasure of severity.

In an exemplary embodiment of the invention, the use of multipleproteins and mRNA expression values provides a robust indicator ofdisease. In some embodiments, only one or two of proteins,phosphorylation and mRNA are used. Optionally, cardiac function values,such as stroke volume are used as well. Optionally, the response oftissue to a field is used as well, for example phosphorylation response.

In an exemplary embodiment of the invention, at least 3, at least 5, atleast 10, at least 20 or more proteins levels, phosphorylation levelsand/or mRNA levels are used to aid in diagnosis. Optionally, a table ismaintained which includes ranges of values that match various diseasetypes and/or conditions. Optionally, as therapy progresses, the patientis re-diagnosed. Optionally, the diagnosis is according to groups ofmRNA and/or proteins that have associated functions.

In an exemplary embodiment of the invention, a DNA chip and/or proteinchip and/or a biochip are used to measure the above levels.

In an exemplary embodiment of the invention, treatments are selected byapplying the above pulses to tissue and measuring the effect.Optionally, a tissue sample is removed, for example by biopsy and arange of sequences are tested on the sample. Optionally, the results ofthe testing indicate which sequence might have a desired therapeuticeffect. Such testing can include analysis and/or application of atreatment, such as an electric field. As noted above, at least some ofthe tests can be meaningfully applied to tissue homogenate and otherunstructured tissue configurations.

In an exemplary embodiment of the invention, different parts of thetissue are tested, for example, to see which tissue part will respondbetter and/or avoid side effects.

Kits

In an exemplary embodiment of the invention, a kit is provided forperforming such analyses as described herein. In an exemplary embodimentof the invention, the kit comprises a set of reagents, antibodies and/orother biochemical tools as known in the art. Alternatively oradditionally, the kit comprises one or more mRNA or protein detectingchips. Alternatively or additionally, the kit comprises software foranalyzing gel migration.

In an exemplary embodiment of the invention, the kit comprises a sourceof treatment, for example electrodes and optionally a power source, forelectrifying a sample for testing its responsiveness. Optionally, thekit includes a sampling means for selecting part of the sample at apre-described time, to check temporal response of the sample. Anexisting kit may be modified for use, for example, a kit available fromBiosite, Inc. to measure blood levels of BNP. Such a kit could includeinstructions for use with the methods described herein and optionallyinclude sampling means or a timer to ensure correct timing ofactivities.

The kit includes or is used with a bioreactor that includes acontrollable sampling element which cans electively extract a portion ofthe sample in the bioreactor and test it in a separate chamber.Optionally, this is embodied using lab-on-chip technology and/or fluidiccontrol of material flow. In an exemplary embodiment of the invention,the controllable sampling element comprises a robot and a pipette thattakes part of the sample and inserts it into an assaying chamber.Various automated assaying devices are known in the art of drugdiscovery and may be used and/or modified to be smaller and/or simpler.

In an exemplary embodiment of the invention, the kit includes a databaseor a link to a database (e.g., on the internet) that links biochemicalmarkers to tissue states, treatment states and/or disease states.

Optionally, the kits are sterile and/or frozen and optionally includeinstructions for use as described herein. Optionally, one or more kitsare packaged with a controller designed for implantation, for use indetermining suitable electrode placement therefore.

Measuring Phosphorylation

In an exemplary embodiment of the invention, the kit includes one ormore antibody reagents useful for detecting phosphorylated and/ordephosphorylated forms of the proteins desired.

Optionally, tracers which are anti-body based are used in-vivo, forexample provided using a catheter or using the delivery electrodes or aseparate needle or other injection mechanism. Optionally, the tracer isradioactive or fluorescent.

A calibration step, for example, per patient, may be carried out.Alternatively or additionally, a comparison before and after fieldapplication is used to determine change in phosphorylation.

Optionally, phosphorylation of proteins which affects ECG signals orcorrelated proteins which affect ECG signals is detected by detectingchanges in ECG signals.

In an exemplary embodiment of the invention, changes in conductionvelocity caused by increased secretion of protein are detected in an ECGmeasurement. Optionally, the measurement is remote from a signalapplication area.

Alternatively or additionally to conduction velocity, changes incontraction velocity under various conditions are assed from images ofthe heart or using other means.

Optionally, other physiological measures are correlated with proteineffects (e.g., in a per-patient or per-population calibration process)and used to estimate effects on protein synthesis from physiologicalmeasures.

Exemplary Cardiac Applications

In an exemplary embodiment of the invention, the above sequences areused to treat tissue plugs that are removed from the body, treated andreinserted. The reinsertion may be, for example, at the location ofremoval, at scarred locations, at locations bordering scars or atotherwise weakened location of the heart.

In an exemplary embodiment of the invention, the above sequences areused as part of a program to re-invigorate scar or fibrotic tissue.Optionally, the effect of the sequence is to cause at least some cellsto become functioning, for example in the border of the scar.

In an exemplary embodiment of the invention, the above sequence is usedto selectively increase oxygen efficiency in some parts of the heart,for example, parts with a vascular blockage thereto.

In an exemplary embodiment of the invention, the above sequences areapplied to tissue transplants, for example, whole heart transplants orplug transplants (from a donor), prior to and/or after transplant.

In an exemplary embodiment of the invention, the above sequences areapplied after DNA or stem cell therapy to the heart, for example, toenhance effects and/or to assist in cellular functional differentiationand/or regeneration.

In an exemplary embodiment of the invention, the above sequences areused to have a desired modeling effect on the heart, for example,modifying an elasticity and/or contractility profile of a heart portionand/or directly controlling conduction velocity.

Three types of remodeling may be provided, possible to different degreesat different parts of the heart. A first type of remodeling is on thebiochemical level and includes, for example, normalization of proteinsynthesis. A second type of remodeling is mechanical remodeling, forexample, fibrosis reduction, chamber size change, vascular increaseand/or wall thickening. A third type of remodeling is functionremodeling which may result form a combination of biochemical andstructural remodeling. This includes, for example, cardiac electricaland/or mechanical activation profile (what gets activated, to whatdegree and in what order), contractility enhancement and conductionvelocity. As described herein, these types of remodeling may be achievedusing methods described herein. Differential remodeling of differenttypes may be achieved, for example, by selectively applying signals witha strong acute effect (which affect mechanical remodeling) and thosewith a long term effect. For example, tissue which is both paced andtreated with non-excitatory signals may thicken, as opposed to tissuewhich is only treated.

In an exemplary embodiment of the invention, multiple methods ofimproving contractility are applied at a same time, or methods ofimproving contractility applied at a same time as methods that reducecontractility such as the initial effect of beta-blockers.

In an exemplary embodiment of the invention, contractility enhancementby effects on membrane proteins is carried out at least partlyindependently from protein effects on SR proteins. Optionally, theselectivity is using methods as described above.

In an exemplary embodiment of the invention, such enhancement isprovided without adversely affecting short term activity, as pacing orinhibition might. In an exemplary embodiment of the invention, theenhancement is local and has fewer systemic effects (as would beexpected with ACE-inhibitors or beta-blockers, for example). In someembodiments, short term activity is improved.

It should be noted that these proteins are also known in other bodyorgans, such as smooth muscle cells and slow-twitch skeletal musclecells. Thus, an electric field as described herein can be used, forexample, to modify PLB phosphorylation in blood vessel cells and/or theGI tract. Optionally, elasticity compliance is restored and vasomotortone is restored and/or responsiveness are restored to blood vesselsand/or GI tract portions using the electric field as described herein.In an exemplary embodiment of the invention, a hardened aorta is mademore supple (and thus relieve cardiac problems) by suitable treatment.Vascular resistance in general, may be modified. Optionally, signals asdescribed herein are used to help increase muscle strength and/ornormalize protein expression therein.

It should be noted that in smooth muscle cells the depolarization cycleis much longer and there is no danger of fatal arrhythmia, so morevaried pulses may be attempted without significant danger and mayprovide longer term effects.

Pulse Optimization

In an exemplary embodiment of the invention, a method of optimizingtreatments to the tissue is provided, taking into account effects onprotein levels. For example, a CCM signal or a pacing signal, exerciseor a pharmaceutical treatment may each (or in combination) have aneffect on protein expression and/or behavior. In an exemplary embodimentof the invention, such a treatment is optimized so that it has a greater(desired) effect on proteins. In one example, a CCM signal is optimizedby applying variations of the signals (e.g., different amplitude,frequencies pulse forms, etc.) to a set of tissue homogenate sets andselecting the signal(s) for which a better effect is achieved. It isnoted that while this may be carried out in vivo, the ability to try outsignals with various pulse parameters on tissue homogenate without theneed for safety testing and worry about danger of damage to apatient/animal, can allow a much faster and/or cheaper search to bemade. Searching methods as known in the art may be used. It is notedthat such searching can also be carried out for small molecule drugswhich have a direct effect on phosphorylation, for example.

It is noted that immediate protein levels may be results that are fasterto achieve or have less noise, than measuring actual improvement in apatient. Thus, protein measurement may allow faster within-patientoptimization and/or allow optimization based on the response to a smallnumber of beats (e.g., 100, 50, 10, 3 or intermediate or fewer numbers),rather than waiting for a longer term effect which may damage the heartif it is negative.

In an exemplary embodiment of the invention, an existing device isoptimized by changing its applied sequence with a sequence selected tohave a desired protein effect.

In an exemplary embodiment of the invention, a device is programmed by aused selecting a desired pulse sequence form an existing set or byselecting parameters which are expected to have a desired proteineffect.

It should be noted that the applied pulse sequence (optionally includingnon-treated beats) and/or desired effect may change over the course of atreatment. One type of change is when the patient state stabilizesand/or the focus of maladaptation changes. In one example, a first stepof treatment is in stabilizing heart treatment and a second step is inincreasing contractility and/or remodeling the heart.

Another type of change is where a different effect is desired atdifferent times during a treatment, for example, a one series of beatsbeing utilized to treat one protein and another series of beats to haveanother effect. It should be noted that not all treatments need to besynchronized the cardiac heart beat.

Both types of change may be controlled using feedback. Optionally, thechange includes one or both of changing the sequence and changing thetissue to which the sequence is applied, for example by switching and/orby moving electrode(s).

In an exemplary embodiment of the invention, the applied sequence takesinto account a provided pharmaceutical and/or a half-life in the bodythereof. In an exemplary embodiment of the invention, a transmitter isprovided to a patient to indicate to the controller that a medicationwas taken.

Optionally, the medication (or another treatment) is provided tospecifically interact with the sequence. For example, a signal ormedicine is provided which has a long effect and while that effect isgoing on, a signal or other treatment is provided which has an oppositeeffect that momentarily counteracts long-term effects. In one example, amedication which extends a refractory period is provided together withan electrical treatment that applies a phosphorylation-modifying signal.In another example, medication is provided to enforce resting of thecells, using a mechanism which does not prevent the CCM or CCM-likesignal from working, possibly, a medication that blocks trans-membranechannels.

It should be appreciated that some sequences that are appliedtherapeutically may change between applications to a same patient, forexample, vary in power, shape, repetition number, duration ofapplication and location. As noted above, such variations may be duringa treatment process of a particular complaint or set of complaints.

General

The following papers, the disclosures of which are incorporated hereinby reference present various results of the effect of a CCM (CardiacContractility Modulation) signal, on gene expression and proteinphosphorylation:

a) an abstract, Control/Tracking Number: 05-A-314176-ACC “ChronicTherapy With Non-Excitatory Cardiac Contractility Modulation ElectricSignals Improves Left Ventricular Function, Reduces Myocardial OxygenConsumption and Increases Myocardial Mechanical Efficiency”, by Hani N.Sabbah, Makoto Imai, Sharad Rastogi, Naveen Sharma, Margaret P.Chandler, Walid Haddad, Yuval Mika, William C. Stanley, Henry FordHealth System, Detroit, Mich., Case Western Reserve University,Cleveland, Ohio; In American College of cardiology foundation.

b) “Non-Excitatory Cardiac Contractility Modulation Electric SignalsNormalize Phosphorylation and Expression of the Sodium Calcium Exchangerin Left Ventricular Myocardium of Dogs with Heart Failure”, by Ramesh C.Gupta, Sudhish Mishra, Sharad Rastogi, Makato Imai, Walid Hadad, YuvalMiKa, Hani N. Sabbah, Henry Ford Health System, Detroit, Mich., ImpulseDynamics, Mount Laurel, N.J.; In Journal of the American College ofCardiology 2005; 45:151A.

c) “Short-Term Therapy with Non-Excitatory Cardiac ContractilityModulation Electric Signals Increases Phosphorylation of Phospholambanin Left Ventricular Myocardium of Dogs With Chronic Heart Failure”, bySudhish Mishra, Ramesh C. Gupta, Sharad Rastogi, Henry Ford HealthSystem, Detroit, Mich.; Walid Haddad, Yuval Mika, Impulse Dynamics USA,Mount Laurel, N.J.; Hani N. Sabbah, Henry Ford Health System, Detroit,Mich.; In Circulation vol. 110; page 111604, 2004.

While the above described apparatus has focused on hardware and/ormethods, it should be understood that the present invention includesprogrammable hardware, software for programmable devices, software forprogramming such hardware and computers including software forprogramming devices. For example, an external programming station may beprovided, which optionally communicates with an implantable device usingtelemetry. Data collection using telemetry may also be practiced. Inaddition, computer readable media including such programs are alsoincluded. Also included are micro-code and other types of programming,as well as hardwired circuitry and ASICs. This is a list of examples andshould not be considered as limiting. An exemplary device/softwareincludes a decision making module, a timing module, a power moduleand/or a signal analysis modules. Section headings are provided fornavigation and should not be considered as limiting their contents tothat section only.

It should be understood that features and/or steps described withrespect to one embodiment may be used with other embodiments and thatnot all embodiments of the invention have all of the features and/orsteps shown in a particular figure or described with respect to one ofthe embodiments. Variations of embodiments described will occur topersons of the art. Furthermore, the terms “comprise,” “include,” “have”and their conjugates, shall mean, when used in the claims, “includingbut not necessarily limited to.” When the term “based on” is used in theclaims it is to be interpreted as meaning “at least partially based on”.

It is noted that some of the above described embodiments may describethe best mode contemplated by the inventors and therefore may includestructure, acts or details of structures and acts that may not beessential to the invention and which are described as examples.Structure and acts described herein are replaceable by equivalents whichperform the same function, even if the structure or acts are different,as known in the art. Therefore, the scope of the invention is limitedonly by the elements and limitations as used in the claims, as issued.

The invention claimed is:
 1. A method of modifying cardiac tissuebehavior at a first location by application of an electric field tocardiac tissue at a second location, comprising: determining a desirednon-acute modification of protein activity and/or gene activity ofcardiac tissue at said first location; selecting electric fieldparameters including one or more of the second location, the duration ofapplication of the field and the power level of the field having anexpected effect of producing said desired non-acute modification of saidprotein activity and/or gene activity at said first location, butwithout causing a significant acute effect at the first location;applying an electric field having the selected parameters to cardiactissue at said second location to produce said selected non-acutemodification at the first location, wherein said first location issufficiently remote from said second location that a significant acuteeffect at the first location does not occur.
 2. A method according toclaim 1, wherein said modification comprises changing a gene program. 3.A method according to claim 2, wherein said gene program comprises afetal gene program.
 4. A method according to claim 1, wherein saidmodification comprises changing a protein activity profile.
 5. A methodaccording to claim 1, wherein the effect of said electric field isallowed to propagate from said second location to said first location bynon-mechanical action.
 6. A method according to claim 1, wherein theeffect of said electric field is allowed to propagate from said secondlocation to said first location in a manner other than by the nervoussystem.
 7. A method according to claim 1, wherein the effect of saidelectric field is allowed to propagate from said second location to saidfirst location by chemical action.
 8. A method according to claim 1,wherein application of said electric field has substantially no longterm effect at said second location.
 9. A method according to claim 1,wherein application of said electric field has a long term effect atsaid second location.
 10. A method according to claim 1, whereinapplication of said electric field has an immediate effect at an area ofsaid second location.
 11. A method according to claim 1, whereinapplication of said electric field has an effect on at least one ofprotein activity and gene activity at said second location.
 12. A methodaccording to claim 1, wherein said first and second locations includecardiac muscle.
 13. A method according to claim 1, wherein said firstlocation is affected to produce said modification within 3 months.
 14. Amethod according to claim 1, wherein said second location is selectedaccording to an expected amount of oxygenation thereat.
 15. A methodaccording to claim 1, comprising selecting said second locationaccording to expected diffusion pathways between said first and secondlocations.
 16. A method according to claim 10, wherein the area of saidsecond location which is immediately affected by said application issmaller than 10 cm².
 17. A method according to claim 1, wherein saidelectric field is applied in a chronic manner.
 18. A method according toclaim 1, wherein applying said electric field increases contractility atsaid second location.
 19. A method according to claim 1, whereinapplying said electric field does not increase oxygen usage at saidsecond location.
 20. A method according to claim 1, wherein saidelectrical field is a non-excitatory field.
 21. A method according toclaim 1, wherein said electrical field reduces contraction at saidsecond location.
 22. A method according to claim 1, further comprisingmodifying said electric field responsive to an effect of said field. 23.A method according to claim 1, wherein said second location is at acardiac septum and said first location is not.
 24. A method according toclaim 1, wherein said applying comprises improving a clinical conditionof a heart failure patient by said applying.
 25. A method according toclaim 10, wherein the area of said second location which is immediatelyaffected by said application is in the range of about 1 to about 20 cm².26. A method according to claim 1, wherein a time scale for an effect onprotein activity at the first location by application of the electricfield at the second location is within the range of about 1 to about 3days.
 27. A method according to claim 1, wherein the effect on geneactivity is a change in mRNA transcription and a time scale for saideffect on mRNA transcription at the first location by a application ofthe electric field at the second location is in the range of about 1 toabout 4 hours.
 28. A method according to claim 1, wherein application ofthe electric field at the second location produces no acute effectsnoticeable at the first location over time scales in the range of about1 to about 20 minutes.
 29. A method according to claim 1, whereinapplication of the electric field at the second location produces noacute effects noticeable at the first location over time scales in therange of about 1 to about 20 hours.
 30. A method according to claim 1,wherein the effect on protein activity is a change in formation ofprotein, and a time scale for said effect on formation of protein at thefirst location by application of the electric field at the secondlocation is within about 3 weeks.